Monoclonal antibodies that specifically bind to matrilin 3 and their use

ABSTRACT

Monoclonal antibodies and antibody fragments that specifically bind to matrilin-3, conjugates including these molecules, and nucleic acid molecules encoding the antibodies, antigen binding fragments and conjugates, are disclosed. Also disclosed are compositions including the disclosed antibodies, antigen binding fragments, conjugates, and nucleic acid molecules. Methods of treating or inhibiting a cartilage disorder in a subject, as well as methods of increasing chondrogenesis in cartilage tissue are further provided. The methods can be used, for example, for treating or inhibiting a growth plate disorder in a subject, such as a skeletal dysplasia or short stature.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of U.S. patent application Ser. No. 17/177,644,filed on Feb. 17, 2021, which is a continuation of U.S. patentapplication Ser. No. 16/391,101, filed on Apr. 22, 2019, issued as U.S.Pat. No. 10,954,291, which is a continuation of U.S. patent applicationSer. No. 15/111,773, filed on Jul. 14, 2016, issued as U.S. Pat. No.10,323,083, which is the U.S. National Stage of InternationalApplication No. PCT/US2015/011433, filed Jan. 14, 2015, and which claimsthe benefit of U.S. Provisional Application No. 61/927,904, filed Jan.15, 2014. The prior applications are all incorporated by referenceherein.

FIELD OF THE DISCLOSURE

This relates to monoclonal antibodies, antigen binding fragments, andconjugates that specifically bind to matrilin-3, as well as methods andagents for treating or preventing cartilage disorders.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing(4239-92095-16_Sequence_Listing.xml, which has a file size of 85,027bytes, and a Date of Creation: May 9, 2023) is herein incorporated byreference in its entirety.

BACKGROUND

A child's growth is dependent on the proper functioning of the growthplate, a specialized cartilage structure located at the end of longbones and within the vertebrae. The primary function of the growth plateis to generate new cartilage, which is then converted into bone tissueand results in the lengthening of bones (i.e., the growth of the child).Mutations in one or more of the many genes that control growth platefunction can cause severe skeletal growth disorders in children.

Current treatments for skeletal cartilage disorders are limited. Onemethod of treatment involves the administration of recombinant growthhormone, but the results are less than optimal and systemic treatmentusing growth hormone carries a risk of increased intracranial pressure,slipped capital femoral epiphysis, insulin resistance, and possibly typeII diabetes mellitus. Thus, there is a need for therapeutic agents andtreatment methods that avoid the systemic risks of current therapies.

SUMMARY

Disclosed herein is the surprising discovery of antibodies and antigenbinding fragments that specifically bind to matrilin-3 and which can beused to target therapeutic payloads to growth plate cartilage.Conjugates of such antibodies or antigen binding fragments that arelinked to a chondrogenic agent target to growth plate cartilage andinduce chondrogenesis, while diminishing adverse effects on non-targettissues, such as those seen with systemic use of growth hormone

Isolated monoclonal antibodies and antigen binding fragments thatspecifically bind to matrilin-3, conjugates thereof, and methods ofusing these molecules, are provided. In some embodiments, the antibodyor antigen binding fragment includes a heavy chain variable regionincluding a heavy chain complementarity determining region (HCDR)1, aHCDR2, and a HCDR3, and a light chain variable region including a lightchain complementarity determining region (LCDR)1, a LCDR2, and a L-CDR3,of the amino acid sequences set forth as one of (a) SEQ ID NO: 1 and SEQID NO: 2, (b) SEQ ID NO: 3 and SEQ ID NO: 4, or (c) SEQ ID NO: 5 and SEQID NO: 6, wherein the monoclonal antibody or antigen binding fragmentspecifically binds to matrilin-3.

In additional embodiments, the HCDR1, HCDR2, and HCDR3 comprise theamino acid sequences set forth as residues 26-33, 51-58, and 97-106 ofSEQ ID NO: 1, respectively, and the LCDR1, LCDR2, and LCDR3 comprise theamino acid sequence set forth as residues 27-32, 50-52, and 89-97 of SEQID NO: 2, respectively. In more embodiments, the HCDR1, HCDR2, and HCDR3comprise the amino acid sequences set forth as residues 26-33, 53-61,and 100-109 of SEQ ID NO: 3, respectively, and the LCDR1, LCDR2, andLCDR3 comprise the amino acid sequence set forth as residues 26-31,49-51, and 88-97 of SEQ ID NO: 4 respectively. In further embodiments,the HCDR1, HCDR2, and HCDR3 comprise the amino acid sequences set forthas residues 26-33, 51-58, 97-108 of SEQ ID NO: 5, respectively, and theLCDR1, LCDR2, and LCDR3 comprise the amino acid sequence set forth asresidues 26-33, 51-53, and 90-100 of SEQ ID NO: 6, respectively.

In some embodiments antibody or antigen binding fragment includes heavyand light chain variable regions including the amino acid sequences setforth as SEQ ID NO: 1 and SEQ ID NO: 2, respectively, SEQ ID NO: 3 andSEQ ID NO: 4, respectively; or SEQ ID NO: 5 and SEQ ID NO: 6,respectively.

In some embodiments, the conjugate can include a chondrogenic agentcovalently linked to the monoclonal antibody or antigen binding fragmentthat specifically binds to matrilin-3. For example, the antibody orantigen binding fragment can be conjugated to a growth hormone, aninsulin-like growth factor-I, an Indian Hedgehog, a bone morphogeneticprotein, a C-type natriuretic protein, a Wnt protein, or a steroid, or afragment thereof that induces chondrogenesis. In additional embodiments,the conjugates can include an anti-arthritis agent, such as aparathyroid (PTH) hormone or functional fragment thereof.

The antibodies, antigen binding fragments, and conjugates can be usedfor a variety of purposes, including in methods of increasingchondrogenesis in cartilage tissue, methods of treating a subject with acartilage disorder, and methods of increasing the height of a subject.These methods can include administering a therapeutically effectiveamount of a disclosed antibody, antigen binding fragment, or conjugateto a subject with a cartilage disorder.

It will be understood that the antibodies, antigen binding fragments andmethods are useful beyond the specific circumstances that are describedin detail herein. The foregoing and features and advantages of thedisclosure will become more apparent from the following detaileddescription, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1D are a series of graphs illustrating selection ofmatrilin-3-binding antibody fragments and assessment of their bindingcharacteristics. (A) A yeast display antibody library was panned againsthuman (first and second rounds of panning) and mouse (third round)matrilin-3 protein. Fluorescence-activated cell sorting showed that thebinding affinity of the enriched pool of yeast display antibodyfragments toward matrilin-3 dramatically increased after three rounds ofselection, when compared to the naïve library. (B-C) The bindingabilities of 36 clones of antibody fragments to human or mouserecombinant matrilin-3 protein were evaluated by ELISA. Plastic wellswere coated with matrilin-3 protein or bovine serum albumin (BSA) andthen incubated with antibody fragments, washed and detected using aHRP-conjugated anti-Fc antibody. Absorbance was normalized to backgroundsignal of 3% non-fat milk (D) The tissue binding specificity of thethree selected clones of antibody fragments were assessed by ELISA.Tissue lysates from a panel of organs from 4-day old mice were used tocoat plastic wells. Individual antibody fragments were incubated in thewells, then washed and detected using HRP-conjugated anti-Fc antibody. Anon-specific antibody fragment directed against an irrelevant proteinserved as a negative control and a commercial antibody againstmatrilin-3 served as a positive control. For panels b-d, data representmean±SEM from three independent experiments (n=9).

FIG. 2 is a set of digital images showing binding of antibody fragmentsto tissues in frozen mouse embryo sections. Antibody fragments 13, 22,and 26 were incubated with frozen E15 mouse embryo sections, thendetected with HRP-conjugated anti-Fc antibody and stained with DABsubstrate to produce a brown color. Immunostaining was observed incartilage tissues, including digits of forepaw (top row) and rib cages(bottom row), but not in non-cartilaginous structures.

FIGS. 3A and 3B are a set of graphs showing assessment of the bindingaffinity of purified matrilin-3-binding antibody fragments. The threeselected antibody fragments, 13, 22 and 26, were expressed and purifiedusing protein A columns. Various concentrations of each antibodyfragment were incubated with recombinant human (A) and mouse (B)matrilin-3 proteins, and binding was measured by ELISA. The negativecontrol (background) lacked an antibody fragment.

FIG. 4 is a graph showing homing of selected matrilin-3-binding antibodyfragments 13, 22 and 26 to cartilage in vivo. Saline, non-specificantibody fragment, or purified antibody fragments were injectedintravenously in 3-week old mice. After 24 hours, various organs werecollected and homogenized, and tissue lysates were used to coat plasticwells. The presence of antibody fragments was detected by ELISA with ananti-Fc antibody. All three antibody fragments were detected incartilage, but not in non-cartilaginous organs. Saline and non-specificantibody served as negative controls. Data represent mean±SEM from fiveindependent experiments.

FIGS. 5A and 5B show a set of schematic diagrams and plasmid vector mapsillustrating construction of dimeric (FIG. 5A) or monomeric (FIG. 5B)fusion proteins including IGF-1 linked to a clone 13-, 22-, or 26-basedscFv and a Fc domain.

FIG. 6 shows a series of graphs illustrating in vitro binding tocartilage by clone 13, 22, or 26 scFvs or corresponding fusion proteinsincluding an IGF-1 domain linked to a clone 13, 22, or 26 scFv and a Fcdomain. ELISA plates were coated with the indicated panel of organlysates and assayed for binding to clone 13, 22, or 26 scFvs or clone13, 22, or 26 scFv-IGF-1-Fc fusion proteins.

FIG. 7 shows a Western blot illustrating that the biological activity ofIGF-1 is not altered by conjugation to a matrilin-3 specific scFv and anFc domain. The IGF-1-induced Erk and Akt phosphorylation in MCF-7 cellswas assayed in the context of IGF-1 alone and the IGF-1-matrilin-3scFv-Fc conjugates using an established assay. MCF-7 cells wereincubated with the IGF-1 alone (10 μM) or IGF-1 conjugated to matrilin-3specific scFV and Fc domains for 30 minutes, lysed, and total Akt andphospho-AKT were measured by Western blot with Akt-specific andphosph-Akt specific antibodies. DMSO was used as solvent control, GAPDHwas used as loading control.

SEQUENCE LISTINGSEQ ID NO: 1 is the amino acid sequence of the V_(H )of the matrilin-3 specific clone 13 mAb.EVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGQGYWFDPWGQGTLVTVSSSEQ ID NO: 2 is the amino acid sequence of the V_(L )of the matrilin-3 specific clone 13 mAb.DVQLTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLTFGGGTKLEIKRSEQ ID NO: 3 is the amino acid sequence of the V_(H )of the matrilin-3 specific clone 22 mAb.QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYGSKWYNDYAPSVKSRISINPDTSKNQFSLQLNSVTPEDTAVYYCTRGIWNAFDIWGQGTMVTVSSSEQ ID NO: 4 is the amino acid sequence of the V_(L )of the matrilin-3 specific clone 22 mAb.SSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPLLVIYDRDNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCQSYDTSLSWVFGGGTQLTVLGSEQ ID NO: 5 is the amino acid sequence of the V_(H )of the matrilin-3 specific clone 26 mAb.EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARWGSGSHAFDIWGQGTTVTVSSSEQ ID NO: 6 is the amino acid sequence of the V_(L )of the matrilin-3 specific clone 26 mAb.SYELTQPPSTSGTPGQRVAISCSGASSNIGSNAVSWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGWVFGGGTQLTVLGSEQ ID NO: 7 is an exemplary nucleic acid sequence encoding an scFv including the V_(H )and V_(L)of the matrilin-3 specific clone 13 mAb.CTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAGGGATCATCCCTATCTTTGGTACAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGAGGCCAAGGGTATTGGTTCGACCCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGAGGTGGCGGGTCTGGTGGAGGCGCTAGCAGTGGTGGCGGATCCGACGTCCAGTTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAATCTCCCGCTCACTTTCGGCGGAGGGACCAAGCTGGAGATCAAASEQ ID NO: 8 is an exemplary nucleic acid sequence encoding an scFv including the V_(H )and V_(L)of the matrilin-3 specific clone 22 mAb.CAGGTACAGCTGCAGCAGTCAGGTCCAGGACTGGTGAAGCCCTCGCAGACCCTCTCACTCACCTGTGCCATCTCCGGGGACAGTGTCTCTAGCAACAGTGCTGCTTGGAACTGGATCAGGCAGTCCCCATCGAGAGGCCTTGAGTGGCTGGGAAGGACATACTACGGGTCCAAGTGGTATAATGATTATGCGCCATCTGTGAAAAGTCGAATAAGTATCAACCCAGACACATCCAAGAACCAGTTCTCCCTGCAACTGAACTCTGTGACTCCCGAAGACACGGCTGTGTATTACTGTACAAGGGGTATTTGGAATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCTTCAGGAGGTGGCGGGTCTGGTGGAGGCGCTAGCGGTGGTGGCGGATCCTCTTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGGATCACATGCCAAGGAGACAGCCTCAGAAGCTATTATGCAAGCTGGTACCAGCAGAAGCCAGGACAGGCCCCTTTACTTGTCATCTATGATAGGGACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGGCTCAGGCGGAAGATGAGGCTGATTATTACTGCCAGTCCTATGACACCAGCCTGAGTTGGGTGTTCGGCGGAGGCACCCAGCTGACCGTCCTCSEQ ID NO: 9 is an exemplary nucleic acid sequence encoding an scFv including the V_(H )and V_(L)of the matrilin-3 specific clone 26 mAb.GAGGTCCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGTCCTCGGTGAAGGTCTCCTGCAAGGCTTCTGGAGGCACCTTCAGCAGCTATGCTATCAGCTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAAGGATCATCCCTATCCTTGGTATAGCAAACTACGCACAGAAGTTCCAGGGCAGAGTCACGATTACCGCGGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGCGAGATGGGGTAGTGGGAGCCATGCTTTTGATATCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAGGAGGTGGCGGGTCTGGTGGAGGCGCTAGCAGTGGTGGCGGATCCTCCTATGAGCTGACTCAGCCACCCTCGACGTCTGGGACCCCCGGGCAGAGGGTCGCCATCTCTTGTTCTGGGGCCAGTTCCAATATCGGAAGTAATGCTGTTAGCTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGCAATAATCAACGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTCAATGGCTGGGTGTTCGGCGGAGGGACCCAGCTCACCGTTTTASEQ ID NO: 10 is the amino acid sequence of the variable region of an scFv including the V_(H)and V_(L )of the matrilin-3 specific clone 13 mAb.LVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGQGYWFDPWGQGTLVTVSSGGGGSGGGASSGGGSDVQLTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLTFGGGTKLEIKSEQ ID NO: 11 is the amino acid sequence of an scFv including the V_(H )and V_(L )of the matrilin-3specific clone 22 mAb.LQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYGSKWYNDYAPSVKSRISINPDTSKNQFSLQLNSVTPEDTAVYYCTRGIWNAFDIWGQGTMVTVSSGGGGSGGGASGGGGSSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPLLVIYDRDNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCQSYDTSLSWVFGGGTQLTVLSEQ ID NO: 12 is the amino acid sequence of an scFv including the V_(H )and V_(L )of the matrilin-3specific clone 26 mAb.LVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARWGSGSHAFDIWGQGTTVTVSSGGGGSGGGASSGGGSSYELTQPPSTSGTPGQRVAISCSGASSNIGSNAVSWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGWVFGGGTQLTVLSEQ ID NO: 13 is an exemplary nucleic acid sequence encoding an scFv including the V_(H )andV_(L )of the matrilin-3 specific clone 13 mAb linked to an Fc domain.ctggtgcagtctggggctgaggtgaagaagcctggggcctcagtgaaggtctcctgcaaggcttctggaggcaccttcagcagctatgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggagggatcatccctatctttggtacagcaaactacgcacagaagttccagggcagagtcacgattaccgcggacaaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgagaggccaagggtattggttcgacccctggggccagggaaccctggtcaccgtctcctcaggaggtggcgggtctggtggaggcgctagcagtggtggcggatccgacgtccagttgacccagtctccatcctccctgtctgcatctgtaggagacagagtcaccatcacttgccaggcgagtcaggacattagcaactatttaaattggtatcagcagaaaccagggaaagcccctaagctcctgatctacgatgcatccaatttggaaacaggggtcccatcaaggttcagtggaagtggatctgggacagattttactttcaccatcagcagcctgcagcctgaagatattgcaacatattactgtcaacagtatgataatctcccgctcactttcggcggagggaccaagctggagatcaaacgtggccaggccggccaagggcccgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctcggggtaaagcggccgctcgaggactaaacgacatcttcgaggctcagaaaatcgaatgcaSEQ ID NO: 14 is an exemplary nucleic acid sequence encoding an scFv including the V_(H )andV_(L )of the matrilin-3 specific clone 22 mAb linked to an Fc domain.caggtacagctgcagcagtcaggtccaggactggtgaagccctcgcagaccctctcactcacctgtgccatctccggggacagtgtctctagcaacagtgctgcttggaactggatcaggcagtccccatcgagaggccttgagtggctgggaaggacatactacgggtccaagtggtataatgattatgcgccatctgtgaaaagtcgaataagtatcaacccagacacatccaagaaccagttctccctgcaactgaactctgtgactcccgaagacacggctgtgtattactgtacaaggggtatttggaatgcttttgatatctggggccaagggacaatggtcaccgtctcttcaggaggtggcgggtctggtggaggcgctagcggtggtggcggatcctcttctgagctgactcaggaccctgctgtgtctgtggccttgggacagacagtcaggatcacatgccaaggagacagcctcagaagctattatgcaagctggtaccagcagaagccaggacaggcccctttacttgtcatctatgatagggacaaccggccctcagggatcccagaccgattctctggctccagctcaggaaacacagcttccttgaccatcactggggctcaggcggaagatgaggctgattattactgccagtcctatgacaccagcctgagttgggtgttcggeggaggcacccagctgaccgtcctcggtggccaggccggccaagggcccgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgactgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggccgctcgaggactaaacgacatcttcgaggctcagaaaatcgaaggcaSEQ ID NO: 15 is an exemplary nucleic acid sequence encoding an scFv including the V_(H )andV_(L )of the matrilin-3 specific clone 26 mAb linked to an Fc domain.gaggtccagctggtgcagtctggggctgaggtgaagaagcctgggtcctcggtgaaggtctcctgcaaggcttctggaggcaccttcagcagctatgctatcagctgggtgcgacaggcccctggacaagggcttgagtggatgggaaggatcatccctatccttggtatagcaaactacgcacagaagttccagggcagagtcacgattaccgcggacaaatccacgagcacagcctacatggagctgagcagcctgagatctgaggacacggccgtgtattactgtgcgagatggggtagtgggagccatgcttttgatatctggggccaagggaccacggtcaccgtctcctcaggaggtggcgggtctggtggaggcgctagcagtggtggcggatcctcctatgagctgactcagccaccctcgacgtctgggacccccgggcagagggtcgccatctcttgttctggggccagttccaatatcggaagtaatgctgttagctggtaccagcagctcccaggaacggcccccaaactcctcatctatagcaataatcaacggccctcaggggtccctgaccgattctctggctccaagtctggcacctcagcctccctggccatcagtgggctccggtccgaggatgaggctgattattactgtgcagcatgggatgacagcctcaatggctgggtgttcggcggagggacccagctcaccgttttaggtggccaggccggccaagggcccgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttcccccccaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaagcggccgctcgaggactaaacgacatcttcgaggctcagaaaatcgaaggcaSEQ ID NO: 16 is the amino acid sequence of the precursor of matrilin-3 (GENBANK Acc. No.NP_002372.1, incorporated by reference herein as present in the database on Dec. 1, 2013)MPRPAPARRLPGLLLLLWPLLLLPSAAPDPVARPGFRRLETRGPGGSPGRRPSPAAPDGAPASGTSEPGRARGAGVCKSRPLDLVFIIDSSRSVRPLEFTKVKTFVSRIIDTLDIGPADTRVAVVNYASTVKIEFQLQAYTDKQSLKQAVGRITPLSTGTMSGLAIQTAMDEAFTVEAGAREPSSNIPKVAIIVTDGRPQDQVNEVAARAQASGIELYAVGVDRADMASLKMMASEPLEEHVFYVETYGVIEKLSSRFQETFCALDPCVLGTHQCQHVCISDGEGKHHCECSQGYTLNADKKTCSALDRCALNTHGCEHICVNDRSGSYHCECYEGYTLNEDRKTCSAQDKCALGTHGCQHICVNDRTGSHHCECYEGYTLNADKKTCSVRDKCALGSHGCQHICVSDGAASYHCDCYPGYTLNEDKKTCSATEEARRLVSTEDACGCEATLAFQDKVSSYLQRLNTKLDDILEKLKINEYGQIHRSEQ ID NO: 17 is an exemplary nucleic acid sequence encoding the precursor of matrilin-3(GENBANK Acc. No. NM_002381.4, incorporated by reference herein as present in the databaseon Dec. 1, 2013).aaatccgagcctcgcgtgggctcctggcccccgacggacaccaccaggcccacggagcccaccatgccgcgcccggcccccgcgcgccgcctcccgggactcctcctgctgctctggccgctgctgctgctgccctccgccgcccccgaccccgtggcccgcccgggcttccggaggctggagacccgaggtcccgggggcagccctggacgccgcccctctcctgcggctcccgacggcgcgcccgcttccgggaccagcgagcctggccgcgcccgcggtgcaggtgtttgcaagagcagacccttggacctggtgtttatcattgatagttctcgtagcgtacggcccctggaattcaccaaagtgaaaacttttgtctcccggataatcgacactctggacattgggccagccgacacgcgggtggcagtggtgaactatgctagcactgtgaagatcgagttccaactccaggcctacacagataagcagtccctgaagcaggccgtgggtcgaatcacacccttgtcaacaggcaccatgtcaggcctagccatccagacagcaatggacgaagccttcacagtggaggcaggggctcgagagccctcttctaacatccctaaggtggccatcattgttacagatgggaggccccaggaccaggtgaatgaggtggcggctcgggcccaagcatctggtattgagctctatgctgtgggcgtggaccgggcagacatggcgtccctcaagatgatggccagtgagcccctagaggagcatgttttctacgtggagacctatggggtcattgagaaactttcctctagattccaggaaaccttctgtgcgctggacccctgtgtgcttggaacacaccagtgccagcacgtctgcatcagtgatggggaaggcaagcaccactgtgagtgtagccaaggatacaccttgaatgccgacaagaaaacgtgttcagccattgtgagtgctatgaaggttataccttgaatgaagacaggaaaacttgttcagctcaagataaatgtgctttgggtacccatgggtgtcagcacatttgtgtgaatgacagaacagggtcccatcattgtgaatgctatgagggctacactctgaatgcagataaaaaaacatgttcagtccgtgacaagtgtgccctaggctctcatggttgccagcacatttgtgtgagtgatggggccgcatcctaccactgtgattgctatcctggctacaccttaaatgaggacaagaaaacatgttcagccactgaggaagcacgaagacttgtttccactgaagatgcttgtggatgtgaagctacactggcattccaggacaaggtcagctcgtatcttcaaagactgaacactaaacttgatgacattttggagaagttgaaaataaatgaatatggacaaatacatcgttaaattgctccaatttctcacctgaaaatgtggacagcttggtgtacttaatactcatgcattcttttgcacacctgttattgccaatgttcctgctaataatttgccattatctgtattaatgcttgaatattactggataaattgtatgaagatcttctgcagaatcagcatgattcttccaaggaaatacatatgcagatacttattaagagcaaactttagtgtctctaagttatgactgtgaaatgattggtaggaaatagaatgaaaagtttagtgtttctttatctactaattgagccatttaatttttaaatgtttatattagataaccatattcacaatggaaactttaggtctagtttcttttgatagtatttataatataaatcaatcttattactgagagtgcaaattgtacaaggtatttacacatacaacttcatataactgagatgaatgtaattttgaactgtttaacactttttgttttttgcttattttgttggagtattattgaagatgtgatcaatagattgtaatacacatatctaaaaatagttaacacagatcaagtgaacattacattgccatttttaattcattctggtctttgaaagaaatgtactactaaagagcactagttgtgaatttagggtgttaaactttttaccaagtacaaaaatcccaaattcactttattattttgcttcaggatccaagtgacaaagttatatatttataaaattgctataaatcgacaaaatctaatgttgtctttttaatgttagtgatccacctgcctcagcctcccaaagtgctgggattacaggcttgaaagtctaacttttttttacttatatatttgatacatataattcttttggctttgaaacttgcaactttgagaacaaaacagtcctttaaattttgcactgctcaattctgtttttcgtttgcattgtctttaatataataaaagttattacctttacatattatcatgtctatttttgatgactcatcaattttgtctattaaagatatttctttaaattaaaaaaaaaaaaaaaaaaSEQ ID NOs: 18-20 are exemplary amino acid and DNA sequences of human growth hormone(GH).SEQ ID NOs: 21-23 and 49 are exemplary amino acid and DNA sequences of IGF-1.SEQ ID NOs: 24-26 are exemplary amino acid and DNA sequences of Indian Hedgehog (IHH)proteins.SEQ ID NOs: 27-29 are exemplary amino acid and DNA sequences of bone morphogenicproteins (BMP).SEQ ID NOs: 30-34 are exemplary amino acid and DNA sequences of CNP proteins.SEQ ID NOs: 35-37 are exemplary amino acid and DNA sequences of WNT proteins.SEQ ID NOs: 38-40 are the amino acid sequences of peptide linkers.SEQ ID NO: 41 is an exemplary amino acid sequence of a fusion protein including IGF-1 linkedto a matrilin-3 clone 13-based scFv linked to a wildtype (dimeric) IgG1-based Fc domain.AGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLGGGGSEVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGQGYWFDPWGQGTLVTVSSGGGGSGGGASSGGGSDVQLTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLTFGGGTKLEIKRGQAGQGPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 42 is an exemplary amino acid sequence of a fusion protein including IGF-1 linkedto a matrilin-3 clone 22-based scFv linked to a wildtype (dimeric) IgG1-based Fc domain.AGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLGGGGSQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYGSKWYNDYAPSVKSRISINPDTSKNQFSLQLNSVTPEDTAVYYCTRGIWNAFDIWGQGTMVTVSSGGGGSGGGASGGGGSSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPLLVIYDRDNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCQSYDTSLSWVFGGGTQLTVLGGQAGQGPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 43 is an exemplary amino acid sequence of a fusion protein including IGF-1 linkedto a matrilin-3 clone 26-based scFv linked to a wildtype (dimeric) IgG1-based Fc domain.AGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLGGGGSEVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARWGSGSHAFDIWGQGTTVTVSSGGGGSGGGASSGGGSSYELTQPPSTSGTPGQRVAISCSGASSNIGSNAVSWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGWVFGGGTQLTVLGGQAGQGPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 44 is an exemplary amino acid sequence of a fusion protein including IGF-1 linkedto a matrilin-3 clone 13-based scFv linked to a mutant (monomeric) IgG1-based Fc domain.AGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLGGGGSGQAGQGPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTSPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKEVQLVQSGAEVKKPGASVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARGQGYWFDPWGQGTLVTVSSGGGGSGGGASSGGGSDVQLTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPLTFGGGTKLEIKRSEQ ID NO: 45 is an exemplary amino acid sequence of a fusion protein including IGF-1 linkedto a matrilin-3 clone 22-based scFv linked to a mutant (monomeric) IgG1-based Fc domain.AGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLGGGGSGQAGQGPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTSPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYGSKWYNDYAPSVKSRISINPDTSKNQFSLQLNSVTPEDTAVYYCTRGIWNAFDIWGQGTMVTVSSGGGGSGGGASGGGGSSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPLLVIYDRDNRPSGIPDRFSGSSSGNTASLTITGAQAEDEADYYCQSYDTSLSWVFGGGTQLTVLGSEQ ID NO: 46 is an exemplary amino acid sequence of a fusion protein including IGF-1 linkedto a matrilin-3 clone 26-based scFv linked to a mutant (monomeric) IgG1-based Fc domain.AGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLGGGGSGQAGQGPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTSPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKEVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRIIPILGIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARWGSGSHAFDIWGQGTTVTVSSGGGGSGGGASSGGGSSYELTQPPSTSGTPGQRVAISCSGASSNIGSNAVSWYQQLPGTAPKLLIYSNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLNGWVFGGGTQLTVLGSEQ ID NO: 47 is an exemplary amino acid sequence of a wildtype IgG1-based Fc domain.DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 48 is an exemplary amino acid sequence of a mutant IgG1-based Fc domain thatdoes not dimerized with other Fc domains.APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTSPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 49 is an exemplary amino acid sequence of a mature IGF-1 polypeptide.SEQ ID NOs: 50-53 are exemplary amino acid and DNA sequences pf parathyroid hormoneproteins.

The nucleic and amino acid sequences are shown using standard letterabbreviations for nucleotide bases, and three letter code for aminoacids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleicacid sequence is shown, but the complementary strand is understood asincluded by any reference to the displayed strand. In the accompanyingsequence listing:

DETAILED DESCRIPTION I. Summary of Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes X, published by Jones & BartlettPublishers, 2009; and Meyers et al. (eds.), The Encyclopedia of CellBiology and Molecular Medicine, published by Wiley-VCH in 16 volumes,2008; and other similar references.

As used herein, the singular forms “a,” “an,” and “the,” refer to boththe singular as well as plural, unless the context clearly indicatesotherwise. For example, the term “an antigen” includes single or pluralantigens and can be considered equivalent to the phrase “at least oneantigen.” As used herein, the term “comprises” means “includes.” Thus,“comprising an antigen” means “including an antigen” without excludingother elements. It is further to be understood that any and all basesizes or amino acid sizes, and all molecular weight or molecular massvalues, given for nucleic acids or polypeptides are approximate, and areprovided for descriptive purposes, unless otherwise indicated. Althoughmany methods and materials similar or equivalent to those describedherein can be used, particular suitable methods and materials aredescribed below. In case of conflict, the present specification,including explanations of terms, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting. To facilitate review of the various embodiments, thefollowing explanations of terms are provided:

Achondroplasia: A cartilage disorder that results in stunted or reducedgrowth, commonly known as Dwarfism. Achondroplasia is usually caused byan autosomal dominant mutation in the gene for fibroblast growth factorreceptor 3 (FGFR-3), which causes an abnormality of cartilage formation.FGFR-3 normally has a negative regulatory effect on chondrocyte growth,and hence bone growth. In achondroplasia, the mutated form of FGFR-3 isconstitutively active, which leads to severely shortened bones. Bothchondrocyte proliferation and differentiation appear to be disturbed,remarkably impairing growth at the growth plate cartilage (P. Krejci etal., J. Cell Sci. 118: 5089-5100 (2005)).

Administration: The introduction of a composition into a subject by achosen route. Administration can be local or systemic. For example, ifthe chosen route is intravenous, the composition is administered byintroducing the composition into a vein of the subject. Exemplary routesof administration include, but are not limited to, oral, injection (suchas subcutaneous, intramuscular, intradermal, intraperitoneal, andintravenous), sublingual, rectal, transdermal (for example, topical),intranasal, vaginal, and inhalation routes. In some examples a discloseda composition that includes a monoclonal antibody or antigen bindingfragment that specifically binds matrilin-3 or conjugate thereof isadministered to a subject.

Agent: Any substance or any combination of substances that is useful forachieving an end or result; for example, a substance or combination ofsubstances useful for decreasing or reducing a cartilage disorder in asubject. Agents include effector molecules and detectable markers. Insome embodiments, the agent is a chondrogenic agent. The skilled artisanwill understand that particular agents may be useful to achieve morethan one result; for example, an agent may be useful as both adetectable marker and a chondrogenic agent.

Antibody: An immunoglobulin, antigen-binding fragment, or derivativethereof, that specifically binds and recognizes an analyte (antigen)such as matrilin-3 or an antigenic fragment of matrilin-3. The term“antibody” is used herein in the broadest sense and encompasses variousantibody structures, including but not limited to monoclonal antibodies,polyclonal antibodies, multispecific antibodies (e.g., bispecificantibodies), and antibody fragments, so long as they exhibit the desiredantigen-binding activity.

Non-limiting examples of antibodies include, for example, intactimmunoglobulins and variants and fragments thereof known in the art thatretain binding affinity for the antigen. Examples of antibody fragmentsinclude but are not limited to Fv, Fab, Fab′, Fab′-SH, F(ab′)₂;diabodies; linear antibodies; single-chain antibody molecules (e.g.scFv); and multispecific antibodies formed from antibody fragments.Antibody fragments include antigen binding fragments either produced bythe modification of whole antibodies or those synthesized de novo usingrecombinant DNA methodologies (see, e.g, Kontermann and Dubel (Ed),Antibody Engineering, Vols. 1-2, 2^(nd) Ed., Springer Press, 2010).

A single-chain antibody (scFv) is a genetically engineered moleculecontaining the V_(H) and V_(L) domains of one or more antibody(ies)linked by a suitable polypeptide linker as a genetically fused singlechain molecule (see, for example, Bird et al., Science, 242:423-426,1988; Huston et al., Proc. Natl. Acad. Sci., 85:5879-5883, 1988; Ahmadet al., Clin. Dev. Immunol., 2012, doi:10.1155/2012/980250; Marbry,IDrugs, 13:543-549, 2010). The intramolecular orientation of theV_(H)-domain and the V_(L)-domain in a scFv, is typically not decisivefor scFvs. Thus, scFvs with both possible arrangements(V_(H)-domain-linker domain-V_(L)-domain; V_(L)-domain-linkerdomain-V_(H)-domain) may be used.

In a dsFv the heavy and light chain variable chains have been mutated tointroduce a disulfide bond to stabilize the association of the chains.Diabodies also are included, which are bivalent, bispecific antibodiesin which V_(H) and V_(L) domains are expressed on a single polypeptidechain, but using a linker that is too short to allow for pairing betweenthe two domains on the same chain, thereby forcing the domains to pairwith complementary domains of another chain and creating two antigenbinding sites (see, for example, Holliger et al., Proc. Natl. Acad.Sci., 90:6444-6448, 1993; Poljak et al., Structure, 2:1121-1123, 1994).

Antibodies also include genetically engineered forms such as chimericantibodies (such as humanized murine antibodies) and heteroconjugateantibodies (such as bispecific antibodies). See also, Pierce Catalog andHandbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J.,Immunology, 3^(rd) Ed., W.H. Freeman & Co., New York, 1997.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. Antibody competition assays are known,and an exemplary competition assay is provided herein.

An antibody may have one or more binding sites. If there is more thanone binding site, the binding sites may be identical to one another ormay be different. For instance, a naturally-occurring immunoglobulin hastwo identical binding sites, a single-chain antibody or Fab fragment hasone binding site, while a bispecific or bifunctional antibody has twodifferent binding sites.

Typically, a naturally occurring immunoglobulin has heavy (H) chains andlight (L) chains interconnected by disulfide bonds. Immunoglobulin genesinclude the kappa, lambda, alpha, gamma, delta, epsilon and mu constantregion genes, as well as the myriad immunoglobulin variable domaingenes. There are two types of light chain, lambda (λ) and kappa (κ).There are five main heavy chain classes (or isotypes) which determinethe functional activity of an antibody molecule: IgM, IgD, IgG, IgA andIgE.

Each heavy and light chain contains a constant region (or constantdomain) and a variable region (or variable domain; see, e.g., Kindt etal. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).) Inseveral embodiments, the heavy and the light chain variable regionscombine to specifically bind the antigen. In additional embodiments,only the heavy chain variable region is required. For example, naturallyoccurring camelid antibodies consisting of a heavy chain only arefunctional and stable in the absence of light chain (see, e.g.,Hamers-Casterman et al., Nature, 363:446-448, 1993; Sheriff et al., Nat.Struct. Biol., 3:733-736, 1996). References to “V_(H)” or “VH” refer tothe variable region of an antibody heavy chain, including that of anantigen binding fragment, such as Fv, scFv, dsFv or Fab. References to“V_(L)” or “VL” refer to the variable domain of an antibody light chain,including that of an Fv, scFv, dsFv or Fab.

Heavy and Light chain variable regions contain a “framework” regioninterrupted by three hypervariable regions, also called“complementarity-determining regions” or “CDRs” (see, e.g., Kabat etal., Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services, 1991). The sequences of the framework regionsof different light or heavy chains are relatively conserved within aspecies. The framework region of an antibody, that is the combinedframework regions of the constituent light and heavy chains, serves toposition and align the CDRs in three-dimensional space.

The CDRs are primarily responsible for binding to an epitope of anantigen. The amino acid sequence boundaries of a given CDR can bereadily determined using any of a number of well-known schemes,including those described by Kabat et al. (“Sequences of Proteins ofImmunological Interest,” 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, M D, 1991; “Kabat” numbering scheme),Al-Lazikani et al., (JMB 273,927-948, 1997; “Chothia” numbering scheme),and Lefranc et al. (“IMGT unique numbering for immunoglobulin and T cellreceptor variable domains and Ig superfamily V-like domains,” Dev. Comp.Immunol., 27:55-77, 2003; “IMGT” numbering scheme). The CDRs of eachchain are typically referred to as CDR1, CDR2, and CDR3 (from theN-terminus to C-terminus), and are also typically identified by thechain in which the particular CDR is located. Thus, a V_(H) CDR3 is theCDR3 from the variable domain of the heavy chain of the antibody inwhich it is found, whereas a V_(L) CDR1 is the CDR1 from the variabledomain of the light chain of the antibody in which it is found. Lightchain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3. Heavychain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3.

A “monoclonal antibody” is an antibody obtained from a population ofsubstantially homogeneous antibodies, that is, the individual antibodiescomprising the population are identical and/or bind the same epitope,except for possible variant antibodies, for example, containingnaturally occurring mutations or arising during production of amonoclonal antibody preparation, such variants generally being presentin minor amounts. In contrast to polyclonal antibody preparations, whichtypically include different antibodies directed against differentdeterminants (epitopes), each monoclonal antibody of a monoclonalantibody preparation is directed against a single determinant on anantigen. Thus, the modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein. In someexamples monoclonal antibodies are isolated from a subject. Monoclonalantibodies can have conservative amino acid substitutions which havesubstantially no effect on antigen binding or other immunoglobulinfunctions. (See, for example, Harlow & Lane, Antibodies, A LaboratoryManual, 2′ ed. Cold Spring Harbor Publications, New York (2013).)

A “humanized” antibody or antigen binding fragment includes a humanframework region and one or more CDRs from a non-human (such as a mouse,rat, or synthetic) antibody or antigen binding fragment. The non-humanantibody or antigen binding fragment providing the CDRs is termed a“donor,” and the human antibody or antigen binding fragment providingthe framework is termed an “acceptor.” In one embodiment, all the CDRsare from the donor immunoglobulin in a humanized immunoglobulin.Constant regions need not be present, but if they are, they can besubstantially identical to human immunoglobulin constant regions, suchas at least about 85-90%, such as about 95% or more identical. Hence,all parts of a humanized antibody or antigen binding fragment, exceptpossibly the CDRs, are substantially identical to corresponding parts ofnatural human antibody sequences.

A “chimeric antibody” is an antibody which includes sequences derivedfrom two different antibodies, which typically are of different species.In some examples, a chimeric antibody includes one or more CDRs and/orframework regions from one human antibody and CDRs and/or frameworkregions from another human antibody.

Anti-arthritis agent: A molecule, such as a chemical compound, smallmolecule, steroid, nucleic acid, polypeptide or other biological agent,that can be used to treat or inhibit arthritis in a subject.Non-limiting examples of anti-arthritis agents include immunosuppressiveagents and anti-inflammatory agents.

Non-limiting examples of anti-arthritis agents that areanti-inflammatory include non-steroidal anti-inflammatory agents (e.g.,ibuprofen, naproxen, COX-2 inhibitors such as celecoxib or rofecoxib)and steroidal agents (e.g, corticosteroids such as dexamethasone,prednisone, and prednisolone). Non-limiting examples of anti-arthritisagents that are immunosuppressive include steroidal agents (e.g,corticosteroids such as dexamethasone, prednisone, and prednisolone),small molecules (e.g., cyclosporine A and F1(506), and biologics (e.g.,TNF inhibitors such as etanercept, anti-CD4 antibodies, anti-TNFαantibodies such as adalimumab and infliximab, anti-CD20 antibodies suchas rituximab, IL-1 receptor antagonists such as anakinra, andparathyroid hormone (PTH) or a fragment thereof that binds to the type 1PTH receptor, such as PTH residues 1-34).

Some anti-arthritis agents exert their effect by reducing inflammationthrough inhibition of the immune system, while other anti-arthritisagents primarily exert their action through non-immune mechanisms, forexample non-steroidal anti-inflammatory drugs that act throughinhibition of inflammatory mediators, such as the cyclooxygenase enzyme.Anti-arthritis agents that inhibit the immune system are often used inthe treatment of autoimmune arthritis. Non-steroidal anti-inflammatorydrugs are often used in the treatment of both autoimmune arthritis andautoimmune arthritis, although either class of drugs can be used toinhibit inflammation in either type of arthritis.

Arthritis: Inflammation of one or more joints in a subject that occursin various forms. Typically, the disease results from mechanical- orautoimmune-induced damage to joint tissue, which causes pain andswelling in affected areas. The joints commonly involved include thehips, knees, lower lumbar and cervical vertebrae, proximal and distalinterphangeal joints of the fingers, first carpometacarpal joints, andfirst tarsometatarsal joints of the feet. Non-limiting types ofarthritis include degenerative arthritis (e.g., osteoarthritis) andautoimmune arthritis (e.g., rheumatoid arthritis).

Rheumatoid arthritis is a chronic, systemic, inflammatory disease thattypically affects the synovial membranes of multiple joints in the body.Because the disease is systemic, there are many extra-articular featuresof the disease as well. For example, neuropathy, scleritis,lymphadenopathy, pericarditis, splenomegaly, arteritis, and rheumatoidnodules are frequent components of the disease. In most cases ofrheumatoid arthritis, the subject has remissions and exacerbations ofthe symptoms. Rheumatoid arthritis considered an autoimmune disease thatis acquired and in which genetic factors appear to play a role.

Osteoarthritis is a degenerative joint disease frequently associatedwith trauma, infection, and/or age-related changes in the affectedjoint. The articular joint structure is degraded, leading to loss ordisruption of joint cartilage and possible bone on bone contact. Thesechanges can result in pain and diminished function. The incidence ofosteoarthritis increases with age, and evidence of osteoarthritisinvolvement can be detected in some joints in the majority of thepopulation by age 65. osteoarthritis is often also accompanied by alocal inflammatory component that may accelerate joint destruction.

Biological sample: A sample obtained from a subject. Biological samplesinclude all clinical samples useful for detection of disease orinfection (for example, a growth disorder) in subjects, including, butnot limited to, cells, tissues, and bodily fluids, such as blood,derivatives and fractions of blood (such as serum), cerebrospinal fluid;as well as biopsied or surgically removed tissue, for example tissuesthat are unfixed, frozen, or fixed in formalin or paraffin. In aparticular example, a biological sample is obtained from a subjecthaving or suspected of having a cartilage disorder; for example, asubject having or suspected of having short stature.

Bone Morphogenic Protein (BMP): A family of proteins belonging to thetransforming growth factor-β superfamily of proteins. BMPs are proteinswhich act to induce the differentiation of mesenchymal-type cells intochondrocytes and osteoblasts before initiating bone formation. Theypromote the differentiation of cartilage- and bone-forming cells nearsites of fractures but also at ectopic locations. In some embodiments, aBMP polypeptide can be conjugated to a matrilin-3 specific antibody orantigen binding fragment. BMPs can promote the conversion of fibroblastsinto chondrocytes and are capable also of inducing the expression of anosteoblast phenotype in non-osteogenic cell types. Exemplary BMP proteinand nucleic acid sequences are known, and further provided herein.

C-type natriuretic protein (CNP): A peptide agonist for the B-typenatriuretic receptor that is known to promote growth platechondrogenesis. In the growth plate, NPR-B is expressed by proliferativecells. In humans, CNP is initially produced from the natriuretic peptideprecursor C(NPPC) gene as a single chain 126-amino acid pre-propolypeptide. Removal of the signal peptide yields pro-CNP, and furthercleavage by the endoprotease furin generates an active 53-amino acidpeptide (CNP-53), which is secreted and enzymatically cleaved to producethe mature 22-amino acid peptide (CNP-22) (Wu, J. Biol. Chem. 278:25847-852 (2003)).

Cartilage: The stiff and inflexible connective tissue found in manyareas in the bodies of humans and other animals, including the jointsbetween bones, the rib cage, the ear, the nose, the elbow, the knee, theankle, the bronchial tubes and the intervertebral discs. Cartilageincludes specialized cells called chondrocytes that produce thecartilage extracellular matrix, which includes a high proportion ofcollagen and proteoglycans. Non-limiting examples of cartilage includegrowth plate cartilage and articular cartilage.

Cartilage disorder: A condition including a cartilage defect in asubject. Cartilage disorders include, for example, growth platecartilage disorders and articulate cartilage disorders. Non-limitingexamples of growth-plate cartilage disorders include skeletal dysplasias(such as achondroplasia, hypochondroplasia, or short stature homeoboxgene (SHOX) deficiency), and short stature, such as idiopathic shortstature, and short stature due to systemic disease (such as systemicinflammatory diseases, renal failure, glucocorticoid therapy, orradiation damage). A non-limiting example of an articulate cartilagedisorder is osteoarthritis.

Several cartilage disorders involve degradation of cartilage over time.Cartilage degradation is the catabolism or the breakdown of thecartilage, including but not limited to degradation of the extracellularmatrix (including collagen, proteoglycans and aggrecans) caused by therelease of abnormally high levels of degradative enzymes by the jointtissues, mainly from articular cartilage and from synovial membranes.Osteoarthritic cartilage degradation is the degradation of cartilage ofthe joints, including but not limited to the elbow, the knee, the handjoints (e.g. wrist, fingers, and thumb), the ankle, the foot, the hip,and the intervertebral discs and cartilage of the growth plate.

Chondrogenic agent: An agent that stimulates or promotes chondrogenesis.Non-limiting examples of chondrogenic agents include growth-regulatingendocrine signaling molecules (such as growth hormone, IGF-1, estrogens,and androgens), growth-regulating paracrine signaling molecules (such asIndian Hedgehog (IHH), bone morphogenetic proteins (BMPs), C-typenatriuretic peptide (CNP), WNTs, and FGFs), and steroids (such asestradiol). In several embodiments, a chondrogenic agent is conjugatedto a matrilin-3 specific antibody or antigen binding fragment.

Chondrocyte: The primary cell type found in healthy cartilage.Chondrocytes produce and maintain the extracellular matrix of cartilage,including collagen and proteoglycans. Methods of identifyingchondrocytes in vitro and in vivo are known, as are methods ofidentifying an increase or decrease in the amount of or number ofchondrocytes.

Chondrogenesis: The production of cartilage. Chondrogenesis can resultfrom, for example, increasing numbers of chondrocytes (proliferation),increasing size of chondrocytes (hypertrophy) and/or production ofcartilage extracellular matrix by chondrocytes, such as an increase incartilage extracellular martrix production by individual chondrocytes,or an increase in the number of chondrocytes producing cartilageextracellular martrix. Methods of detecting and/or quantifyingchondrogenesis are known and further described herein Some of thetherapeutic agents disclosed herein for treating cartilage disordersexert their therapeutic effect by stimulating chondrogenesis. The effectof stimulating chondrogenesis means a stimulation of genesis of acartilage tissue, particularly the cartilage tissue at the epiphysisregion, and is an action which also includes functional maintenance ofcartilage tissues. Some of the actions are effected via any one ofstimulating chondrocyte growth, stimulating chondrocyte differentiation,inhibiting cartilage calcification or inhibiting cartilage degradation,or a multiple combination thereof.

Conditions sufficient to form an immune complex: Conditions which allowan antibody or antigen binding fragment thereof to bind to its cognateepitope to a detectably greater degree than, and/or to the substantialexclusion of, binding to substantially all other epitopes. Conditionssufficient to form an immune complex are dependent upon the format ofthe binding reaction and typically are those utilized in immunoassayprotocols or those conditions encountered in vivo. See Harlow & Lane,Antibodies, A Laboratory Manual, 2′ ed. Cold Spring Harbor Publications,New York (2013) for a description of immunoassay formats and conditions.The conditions employed in the methods are “physiological conditions”which include reference to conditions (e.g., temperature, osmolarity,pH) that are typical inside a living mammal or a mammalian cell. Whileit is recognized that some organs are subject to extreme conditions, theintra-organismal and intracellular environment normally lies around pH 7(e.g., from pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), containswater as the predominant solvent, and exists at a temperature above 0°C. and below 50° C. Osmolarity is within the range that is supportive ofcell viability and proliferation.

The formation of an immune complex can be detected through conventionalmethods known to the skilled artisan, for instance immunohistochemistry,immunoprecipitation, flow cytometry, immunofluorescence microscopy,ELISA, immunoblotting (for example, Western blot), magnetic resonanceimaging, CT scans, X-ray and affinity chromatography. Immunologicalbinding properties of selected antibodies may be quantified usingmethods well known in the art.

Conjugate: A complex of two molecules linked together, for example,linked together by a covalent bond. In one embodiment, an antibody islinked to an effector molecule; for example, an antibody thatspecifically binds to matrilin-3 covalently linked to an effectormolecule. The linkage can be by chemical or recombinant means. In oneembodiment, the linkage is chemical, wherein a reaction between theantibody moiety and the effector molecule has produced a covalent bondformed between the two molecules to form one molecule. A peptide linker(short peptide sequence) can optionally be included between the antibodyand the effector molecule. Because conjugates can be prepared from twomolecules with separate functionalities, such as an antibody and aneffector molecule, they are also sometimes referred to as “chimericmolecules.”

Conservative variants: “Conservative” amino acid substitutions are thosesubstitutions that do not substantially alter the biological function ofa protein, such as substitutions that do not substantially decrease thebinding affinity of an antibody for an antigen (for example, the bindingaffinity of an antibody for matrilin-3). For example, a human antibodythat specifically binds matrilin-3 can include at most about 1, at mostabout 2, at most about 5, at most about 10, or at most about 15conservative substitutions and specifically bind the matrilin-3polypeptide. The term conservative variation also includes the use of asubstituted amino acid in place of an unsubstituted parent amino acid,provided that antibody retains binding affinity for matrilin-3.

Conservative amino acid substitution tables providing functionallysimilar amino acids are well known to one of ordinary skill in the art.The following six groups are examples of amino acids that are consideredto be conservative substitutions for one another:

-   -   1) Alanine (A), Serine (S), Threonine (T);    -   2) Aspartic acid (D), Glutamic acid (E);    -   3) Asparagine (N), Glutamine (Q);    -   4) Arginine (R), Lysine (K);    -   5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and    -   6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Non-conservative substitutions are those that reduce an activity orfunction of the matrilin-3-specific antibody, such as the ability tospecifically bind to matrilin-3. For instance, if an amino acid residueis essential for a function of the protein, even an otherwiseconservative substitution may disrupt that activity. Thus, aconservative substitution does not alter the basic function of a proteinof interest.

Contacting: Placement in direct physical association; includes both insolid and liquid form, which can take place either in vivo or in vitro.Contacting includes contact between one molecule and another molecule,for example the amino acid on the surface of one polypeptide, such as anantigen, that contacts another polypeptide, such as an antibody.Contacting can also include contacting a cell for example by placing anantibody in direct physical association with a cell.

Control: A reference standard. In some embodiments, the control is anegative control, such as tissue sample obtained from a patient thatdoes not have a cartilage disorder, or a tissue sample from a tissuethat is not affected by the cartilage disorder. In other embodiments,the control is a positive control, such as a tissue sample obtained froma patient diagnosed with a cartilage disorder, or a tissue sample from atissue affected by the cartilage disorder. In still other embodiments,the control is a historical control or standard reference value or rangeof values (such as a previously tested control sample, such as fromgroup of cartilage disorder patients with known prognosis or outcome, orgroup of samples that represent baseline or normal values).

A difference between a test sample and a control can be an increase orconversely a decrease. The difference can be a qualitative difference ora quantitative difference, for example a statistically significantdifference. In some examples, a difference is an increase or decrease,relative to a control, of at least about 5%, such as at least about 10%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 100%, at least about 150%, at leastabout 200%, at least about 250%, at least about 300%, at least about350%, at least about 400%, or at least about 500%.

Degenerate variant: In the context of the present disclosure, a“degenerate variant” refers to a polynucleotide encoding a protein (forexample, an antibody that specifically binds matrilin-3) that includes asequence that is degenerate as a result of the genetic code. There aretwenty natural amino acids, most of which are specified by more than onecodon. Therefore, all degenerate nucleotide sequences are included aslong as the amino acid sequence of the antibody that binds matrilin-3encoded by the nucleotide sequence is unchanged.

Detectable marker: A detectable molecule (also known as a label) that isconjugated directly or indirectly to a second molecule, such as anantibody, to facilitate detection of the second molecule. For example,the detectable marker can be capable of detection by ELISA,spectrophotometry, flow cytometry, microscopy or diagnostic imagingtechniques (such as CT scans, MRIs, ultrasound, fiberoptic examination,and laparoscopic examination). Specific, non-limiting examples ofdetectable markers include fluorophores, chemiluminescent agents,enzymatic linkages, radioactive isotopes and heavy metals or compounds(for example super paramagnetic iron oxide nanocrystals for detection byMRI). In one example, a “labeled antibody” refers to incorporation ofanother molecule in the antibody. For example, the label is a detectablemarker, such as the incorporation of a radiolabeled amino acid orattachment to a polypeptide of biotinyl moieties that can be detected bymarked avidin (for example, streptavidin containing a fluorescent markeror enzymatic activity that can be detected by optical or colorimetricmethods). Various methods of labeling polypeptides and glycoproteins areknown in the art and may be used. Examples of labels for polypeptidesinclude, but are not limited to, the following: radioisotopes orradionuclides (such as ³⁵S or ¹³¹I) fluorescent labels (such asfluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors),enzymatic labels (such as horseradish peroxidase, beta-galactosidase,luciferase, alkaline phosphatase), chemiluminescent markers, biotinylgroups, predetermined polypeptide epitopes recognized by a secondaryreporter (such as a leucine zipper pair sequences, binding sites forsecondary antibodies, metal binding domains, epitope tags), or magneticagents, such as gadolinium chelates. In some embodiments, labels areattached by spacer arms of various lengths to reduce potential sterichindrance. Methods for using detectable markers and guidance in thechoice of detectable markers appropriate for various purposes arediscussed for example in Sambrook et al. (Molecular Cloning: ALaboratory Manual, 4^(th) ed, Cold Spring Harbor, New York, 2012) andAusubel et al. (In Current Protocols in Molecular Biology, John Wiley &Sons, New York, through supplement 104, 2013).

Detecting: To identify the existence, presence, or fact of something.General methods of detecting are known to the skilled artisan and may besupplemented with the protocols and reagents disclosed herein. Forexample, included herein are methods of detecting cartilage, such asgrowth-plate cartilage, in a subject.

Effector molecule: A molecule intended to have or produce a desiredeffect; for example, a desired effect on a cell to which the effectormolecule is targeted. Effector molecules can include, for example,polypeptides and small molecules. In one non-limiting example, theeffector molecule is a chondrogenic agent. The skilled artisan willunderstand that some effector molecules may have or produce more thanone desired effect. In one example, an effector molecule is the portionof a chimeric molecule, for example a chimeric molecule that includes adisclosed antibody or fragment thereof, that is intended to have adesired effect on a cell or tissue to which the chimeric molecule istargeted.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic, i.e. that elicita specific immune response. An antibody specifically binds a particularantigenic epitope on a polypeptide. In some examples a disclosedantibody specifically binds to an epitope on matrilin-3.

Expressed: Translation of a nucleic acid into a protein. Proteins may beexpressed and remain intracellular, become a component of the cellsurface membrane, or be secreted into the extracellular matrix ormedium.

Expression Control Sequences: Nucleic acid sequences that regulate theexpression of a heterologous nucleic acid sequence to which it isoperatively linked. Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus expression control sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalfor introns, maintenance of the correct reading frame of that gene topermit proper translation of mRNA, and stop codons. The term “controlsequences” is intended to include, at a minimum, components whosepresence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Expression control sequences can include apromoter.

A promoter is a minimal sequence sufficient to direct transcription.Also included are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ regions of the gene. Bothconstitutive and inducible promoters are included (see for example,Bitter et al., Methods in Enzymology 153:516-544, 1987). For example,when cloning in bacterial systems, inducible promoters such as pL ofbacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) andthe like may be used. In one embodiment, when cloning in mammalian cellsystems, promoters derived from the genome of mammalian cells (such asmetallothionein promoter) or from mammalian viruses (such as theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) can be used. Promoters produced byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the nucleic acid sequences. A polynucleotide can beinserted into an expression vector that contains a promoter sequencewhich facilitates the efficient transcription of the inserted geneticsequence of the host. The expression vector typically contains an originof replication, a promoter, as well as specific nucleic acid sequencesthat allow phenotypic selection of the transformed cells.

Expression vector: A vector comprising a recombinant polynucleotidecomprising expression control sequences operatively linked to anucleotide sequence to be expressed. An expression vector comprisessufficient cis-acting elements for expression; other elements forexpression can be supplied by the host cell or in an in vitro expressionsystem. Expression vectors include all those known in the art, such ascosmids, plasmids (e.g., naked or contained in liposomes) and viruses(e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associatedviruses) that incorporate the recombinant polynucleotide.

Fc polypeptide: The polypeptide including the constant region of anantibody excluding the first constant region immunoglobulin domain. Fcregion generally refers to the last two constant region immunoglobulindomains of IgA, IgD, and IgG, and the last three constant regionimmunoglobulin domains of IgE and IgM. An Fc region may also includepart or all of the flexible hinge N-terminal to these domains. For IgAand IgM, an Fc region may or may not include the tailpiece, and may ormay not be bound by the J chain. For IgG, the Fc region includesimmunoglobulin domains Cgamma2 and Cgamma3 (Cγ2 and Cγ3) and the lowerpart of the hinge between Cgamma1 (Cγ1) and Cγ2. Although the boundariesof the Fc region may vary, the human IgG heavy chain Fc region isusually defined to include residues C226 or P230 to itscarboxyl-terminus, wherein the numbering is according to the EU index asin Kabat. For IgA, the Fc region includes immunoglobulin domains Calpha2and Calpha3 (Cα2 and Cα3) and the lower part of the hinge betweenCalpha1 (Cal) and Cα2. In some embodiments, the Fc can be a mutant IgGFc domain that does not dimerize under physiological conditions, forexample, an Fc domain comprising the amino acid sequence set forth asSEQ ID NO: 48.

Framework Region: Amino acid sequences interposed between CDRs in aheavy or light variable region of an antibody. Includes variable lightand variable heavy framework regions. The framework regions serve tohold the CDRs in an appropriate orientation.

Growth Plate: A specialized cartilage structure present near the ends oftubular bones and vertebrae, and which includes chondrocytes thatproduce cartilage extracellular matrix. The primary function of thegrowth plate is to generate new cartilage, which is then remodeled intobone tissue, resulting in bone elongation. Because body length islargely determined by the lengths of long bones and vertebrae, boneformation at the growth plates is the underlying mechanism responsiblefor increasing height during childhood. In addition to geneticdisorders, acquired endocrine, nutritional, or inflammatory disordersalso impair bone growth at the growth plate, resulting in short stature.Some of the more common acquired conditions that inhibit childhoodgrowth include renal failure, inflammatory bowel disease, glucocorticoidtherapy such as prednisone, or radiation therapy.

There are three principal zones of the growth plate: resting,proliferative, and hypertrophic. In normal endochondral bone growth,chondrocytes organize in columns and proliferate in the proliferativezone of the growth plate. The hypertrophic zone is where the cellsbecome large and eventually undergo apoptosis (programmed cell death).The hypertrophic zone is invaded by blood vessels, osteoclasts, anddifferentiating osteoblasts which remodel the newly formed cartilageinto bone tissue. Impaired proliferation, differentiation, or functionof the growth plate chondrocytes in these zones of the growth plate canlead to a growth plate cartilage disorder, for example, achondroplasia.

Longitudinal bone growth at the growth plate is a complex process whichrequires multiple intracellular, endocrine, and paracrine pathways tofunction normally. Consequently, mutations in hundreds of genes that arerequired for growth plate function give rise to disorders of skeletalgrowth, including skeletal dysplasias, in which the bones are typicallyshort and malformed, often causing major disability.

Increase: To increase the quality, amount, or strength of something; forexample an increase in the size or volume of cartilage (such as growthplate cartilage or articulate cartilage), to increase the number oramount or production of chondrocytes in cartilage tissue (such as growthplate or articular cartilage). In a particular example, a therapyincreases in the size or volume of cartilage (such as growth platecartilage or articulate cartilage), the number or amount or productionof chondrocytes in cartilage tissue (such as growth plate or articularcartilage), and/or the linear growth of a subject, subsequent to thetherapy, such as an increase of at least 10%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, or at least 90%. Such increases can be measured using known methodsand those disclosed herein.

Isolated: A biological component (such as a nucleic acid, peptide,protein or protein complex, for example an antibody) that has beensubstantially separated, produced apart from, or purified away fromother biological components in the cell of the organism in which thecomponent naturally occurs, that is, other chromosomal andextra-chromosomal DNA and RNA, and proteins. Thus, isolated nucleicacids, peptides and proteins include nucleic acids and proteins purifiedby standard purification methods. The term also embraces nucleic acids,peptides and proteins prepared by recombinant expression in a host cell,as well as, chemically synthesized nucleic acids. A isolated nucleicacid, peptide or protein, for example an antibody, can be at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% pure.

K_(D): The dissociation constant for a given interaction, such as apolypeptide ligand interaction or an antibody antigen interaction. Forexample, for the bimolecular interaction of an antibody or antigenbinding fragment (such as a matrilin-3 specific antibody or conjugatethereof) and an antigen (such as matrilin-3 protein) it is theconcentration of the individual components of the bimolecularinteraction divided by the concentration of the complex.

Linear Growth: An increase in the long bones of a subject. In humans, anincrease in linear growth correlates with an increase in height, and canbe identified, for example, by detecting an increase in the height of ahuman subject over time. In several non-human subjects (e.g., murinesubjects), an increase in linear growth can be identified by detectingan increase in the length of a long bone in the subject (e.g., thefemur) or in body length over time.

Linker: A bi-functional molecule that can be used to link two moleculesinto one contiguous molecule, for example, to link an effector moleculeto an antibody. In some embodiments, the provided conjugates include alinker between the effector molecule or detectable marker and anantibody. In some cases, a linker is a peptide within an antigen bindingfragment (such as an Fv fragment) which serves to indirectly bond thevariable heavy chain to the variable light chain. Non-limiting examplesof peptide linkers include a (GGGGS)₁ (SEQ ID NO: 38), (GGGGS)₂ (SEQ IDNO: 39), or a (GGGGS)₃ (SEQ ID NO: 40) linker.

The terms “conjugating,” “joining,” “bonding,” or “linking” can refer tomaking two molecules into one contiguous molecule; for example, linkingtwo polypeptides into one contiguous polypeptide, or covalentlyattaching an effector molecule or detectable marker radionuclide orother molecule to a polypeptide, such as an scFv. In the specificcontext, the terms include reference to joining a ligand, such as anantibody moiety, to an effector molecule. The linkage can be either bychemical or recombinant means. “Chemical means” refers to a reactionbetween the antibody moiety and the effector molecule such that there isa covalent bond formed between the two molecules to form one molecule.

Matrilin-3: A member of the non-collagenous matrilin family ofextracellular matrix proteins that share common domains and similarfunctions. Matrilin-3 is expressed primarily in cartilage and mutationsin the gene encoding matrilin-3 protein can result in a variety ofskeletal diseases including chondrodysplasia and osteoarthritis.Exemplary protein and nucleic acid sequences for human matrilin-3precursor are set forth as SEQ ID NOs: 16 and 17, respectively (NCBIAcc. Nos. NP_002372.1 and NM_002381.4, incorporated by reference hereinas present in the database on Dec. 1, 2013).

Nucleic acid: A polymer composed of nucleotide units (ribonucleotides,deoxyribonucleotides, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof) linked viaphosphodiester bonds, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof. Thus, the termincludes nucleotide polymers in which the nucleotides and the linkagesbetween them include non-naturally occurring synthetic analogs, such as,for example and without limitation, phosphorothioates, phosphoramidates,methyl phosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like. Suchpolynucleotides can be synthesized, for example, using an automated DNAsynthesizer. The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 50 nucleotides. It willbe understood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e.,A, U, G, C) in which “U” replaces “T.”

Conventional notation is used herein to describe nucleotide sequences:the left-hand end of a single-stranded nucleotide sequence is the5′-end; the left-hand direction of a double-stranded nucleotide sequenceis referred to as the 5′-direction. The direction of 5′ to 3′ additionof nucleotides to nascent RNA transcripts is referred to as thetranscription direction. The DNA strand having the same sequence as anmRNA is referred to as the “coding strand;” sequences on the DNA strandhaving the same sequence as an mRNA transcribed from that DNA and whichare located 5′ to the 5′-end of the RNA transcript are referred to as“upstream sequences;” sequences on the DNA strand having the samesequence as the RNA and which are 3′ to the 3′ end of the coding RNAtranscript are referred to as “downstream sequences.”

“cDNA” refers to a DNA that is complementary or identical to an mRNA, ineither single stranded or double stranded form.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA produced by that geneproduces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and non-codingstrand, used as the template for transcription, of a gene or cDNA can bereferred to as encoding the protein or other product of that gene orcDNA. Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons.

A polynucleotide or nucleic acid sequence refers to a polymeric form ofnucleotide at least 10 bases in length. A recombinant polynucleotideincludes a polynucleotide that is not immediately contiguous with bothof the coding sequences with which it is immediately contiguous (one onthe 5′ end and one on the 3′ end) in the naturally occurring genome ofthe organism from which it is derived. The term therefore includes, forexample, a recombinant DNA which is incorporated into a vector; into anautonomously replicating plasmid or virus; or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g., acDNA) independent of other sequences. The nucleotides can beribonucleotides, deoxyribonucleotides, or modified forms of eithernucleotide. The term includes single- and double-stranded forms of DNA.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter, such as the CMV promoter, isoperably linked to a coding sequence if the promoter affects thetranscription or expression of the coding sequence. Generally, operablylinked DNA sequences are contiguous and, where necessary to join twoprotein-coding regions, in the same reading frame.

Osteoarthritis: A disorder caused by gradual degradation of articularcartilage with inadequate repair, which affects 30-50% of older adults.In patients with osteoarthritis, the cartilage extracellular matrixproteins, which are the functional entity of the cartilage, are reduced,and the number of chondrocytes decreases (Aigner and Kim, Arth. Rheum.46(8): 1986-1996 (2002)). Methods of identifying a subject withosteoarthritis are known; see, e.g., Moskowitz et al. (Eds),Osteoarthritis: Diagnosis and Medical/Surgical Management, LippincottWilliams & Wilkins; Fourth edition, 2006.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers provided herein are conventional. Remington's PharmaceuticalSciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition(1975), describes compositions and formulations suitable forpharmaceutical delivery of the fusion proteins herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually include injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (for example, powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch, ormagnesium stearate. In addition to biologically-neutral carriers,pharmaceutical compositions to be administered can contain minor amountsof non-toxic auxiliary substances, such as wetting or emulsifyingagents, added preservatives (such as non-natural preservatives), and pHbuffering agents and the like, for example sodium acetate or sorbitanmonolaurate. In particular examples, the pharmaceutically acceptablecarrier is sterile and suitable for parenteral administration, forexample by injection. In some embodiments, the active agent andpharmaceutically acceptable carrier are provided in a unit dosage formsuch as a pill or in a selected quantity in a vial. Unit dosage formscan include one dosage or multiple dosages (for example, in a vial fromwhich metered dosages of the agents can selectively be dispensed).

Polypeptide: Any chain of amino acids, regardless of length orpost-translational modification (e.g., glycosylation orphosphorylation). In one embodiment, the polypeptide is a disclosedantibody or a fragment thereof. A “residue” refers to an amino acid oramino acid mimetic incorporated in a polypeptide by an amide bond oramide bond mimetic. A polypeptide has an amino terminal (N-terminal) endand a carboxy terminal (C-terminal) end.

Polypeptide modifications: Peptides can be modified by a variety ofchemical techniques to produce derivatives having essentially the sameactivity and conformation as the unmodified peptides, and optionallyhaving other desirable properties. For example, carboxylic acid groupsof the protein, whether carboxyl-terminal or side chain, may be providedin the form of a salt of a pharmaceutically-acceptable cation oresterified to form a C₁-C₁₆ ester, or converted to an amide of formulaNR₁R₂ wherein R₁ and R₂ are each independently H or C₁-C₁₆ alkyl, orcombined to form a heterocyclic ring, such as a 5- or 6-membered ringAmino groups of the peptide, whether amino-terminal or side chain, maybe in the form of a pharmaceutically-acceptable acid addition salt, suchas the HCl, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric andother organic salts, or may be modified to C₁-C₁₆ alkyl or dialkyl aminoor further converted to an amide.

Hydroxyl groups of the peptide side chains can be converted to C₁-C₁₆alkoxy or to a C₁-C₁₆ ester using well-recognized techniques. Phenyl andphenolic rings of the peptide side chains can be substituted with one ormore halogen atoms, such as F, Cl, Br or I, or with C₁-C₁₆ alkyl, C₁-C₁₆alkoxy, carboxylic acids and esters thereof, or amides of suchcarboxylic acids. Methylene groups of the peptide side chains can beextended to homologous C₂-C₄ alkylenes. Thiols can be protected with anyone of a number of well-recognized protecting groups, such as acetamidegroups.

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified peptidepreparation is one in which the peptide or protein (such as an antibody)is more enriched than the peptide or protein is in its naturalenvironment within a cell. In one embodiment, a preparation is purifiedsuch that the protein or peptide represents at least 50% of the totalpeptide or protein content of the preparation, such as at least 80%, atleast 90%, at least 95% or greater of the total peptide or proteincontent.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination can be accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, for example, by genetic engineering techniques. Arecombinant protein is a protein encoded by a heterologous (for example,recombinant) nucleic acid that has been introduced into a host cell,such as a bacterial or eukaryotic cell. The nucleic acid can beintroduced, for example, on an expression vector having signals capableof expressing the protein encoded by the introduced nucleic acid or thenucleic acid can be integrated into the host cell chromosome.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Homologs or variants of a polypeptide will possess a relatively highdegree of sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci.U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins andSharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents adetailed consideration of sequence alignment methods and homologycalculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, MD)and on the internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet.

Homologs and variants of a V_(L) or a V_(H) of an antibody thatspecifically binds a polypeptide are typically characterized bypossession of at least about 75%, for example at least about 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identitycounted over the full length alignment with the amino acid sequence ofinterest. Proteins with even greater similarity to the referencesequences will show increasing percentage identities when assessed bythis method, such as at least 80%, at least 85%, at least 90%, at least95%, at least 98%, or at least 99% sequence identity. When less than theentire sequence is being compared for sequence identity, homologs andvariants will typically possess at least 80% sequence identity overshort windows of 10-20 amino acids, and may possess sequence identitiesof at least 85% or at least 90% or 95% depending on their similarity tothe reference sequence. Methods for determining sequence identity oversuch short windows are available at the NCBI website on the internet.One of skill in the art will appreciate that these sequence identityranges are provided for guidance only; it is entirely possible thatstrongly significant homologs could be obtained that fall outside of theranges provided.

Terms used to describe sequence relationships between two or morenucleotide sequences or amino acid sequences include “referencesequence,” “selected from,” “comparison window,” “identical,”“percentage of sequence identity,” “substantially identical,”“complementary,” and “substantially complementary.”

For sequence comparison of nucleic acid sequences, typically onesequence acts as a reference sequence, to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are entered into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. Default program parameters are used. Methods of alignment ofsequences for comparison are well known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.48:443, 1970, by the search for similarity method of Pearson & Lipman,Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, WI), or by manual alignment and visual inspection(see, e.g., Sambrook et al. (Molecular Cloning: A Laboratory Manual,4^(th) ed, Cold Spring Harbor, New York, 2012) and Ausubel et al. (InCurrent Protocols in Molecular Biology, John Wiley & Sons, New York,through supplement 104, 2013). One example of a useful algorithm isPILEUP. PILEUP uses a simplification of the progressive alignment methodof Feng & Doolittle, J. Mol. Evol. 35:351-360, 1987. The method used issimilar to the method described by Higgins & Sharp, CABIOS 5:151-153,1989. Using PILEUP, a reference sequence is compared to other testsequences to determine the percent sequence identity relationship usingthe following parameters: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps. PILEUP can be obtained from theGCG sequence analysis software package, e.g., version 7.0 (Devereaux etal., Nuc. Acids Res. 12:387-395, 1984.

Another example of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and the BLAST2.0 algorithm, which are described in Altschul et al., J. Mol. Biol.215:403-410, 1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402,1977. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information(ncbi.nlm.nih.gov). The BLASTN program (for nucleotide sequences) usesas defaults a word length (W) of 11, alignments (B) of 50, expectation(E) of 10, M=5, N=−4, and a comparison of both strands. The BLASTPprogram (for amino acid sequences) uses as defaults a word length (W) of3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989). Anoligonucleotide is a linear polynucleotide sequence of up to about 100nucleotide bases in length.

Short stature: A height that is below the normal range either duringchildhood or adulthood. In some embodiments, a subject with shortstature is one with a height that is less than two standard deviationsbelow the mean height for comparable age and sex. The person of ordinaryskill in the art can readily determine if a particular subject is asubject with short stature.

Specifically bind: When referring to an antibody or antigen bindingfragment thereof, refers to a binding reaction which determines thepresence of a target protein, peptide, or polysaccharide in the presenceof a heterogeneous population of proteins and other biologics. Thus,under designated conditions, an antibody binds preferentially to aparticular target protein, peptide or polysaccharide (such as an epitopeof matrilin-3) and does not bind in a significant amount to otherproteins or polysaccharides present in the sample or subject. Specificbinding can be determined by methods known in the art. With reference toan antibody antigen complex, specific binding of the antigen andantibody has a K_(d) of less than about 10⁻⁷ Molar (M), such as lessthan about 10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, or even less than about 10⁻¹¹ M.

Particular antibodies disclosed herein specifically bind only to adefined target (or multiple targets, in the case of a bispecificantibody). Thus, an antibody that specifically binds to matrilin-3 is anantibody that binds substantially to matrilin-3, including cells ortissue expressing matrilin-3, substrate to which the matrilin-3 isattached, or matrilin-3 in a biological specimen. It is, of course,recognized that a certain degree of non-specific interaction may occurbetween an antibody or conjugate including an antibody (such as anantibody that specifically binds matrilin-3 or conjugate including suchantibody) and a non-target (such as a cell that does not expressmatrilin-3). Typically, specific binding results in a much strongerassociation between the antibody and protein or cells bearing theantigen than between the antibody and protein or cells lacking theantigen. Specific binding typically results in greater than 2-fold, suchas greater than 5-fold, greater than 10-fold, or greater than 100-foldincrease in amount of bound antibody (per unit time) to a proteinincluding the epitope or cell or tissue expressing the target epitope ascompared to a protein or cell or tissue lacking this epitope. Specificbinding to a protein under such conditions requires an antibody that isselected for its specificity for a particular protein. A variety ofimmunoassay formats are appropriate for selecting antibodies or otherligands specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. SeeHarlow & Lane, Antibodies, A Laboratory Manual, 2^(nd) ed., Cold SpringHarbor Publications, New York (2013), for a description of immunoassayformats and conditions that can be used to determine specificimmunoreactivity.

Subject: Any mammal, such as humans, non-human primates, pigs, sheep,cows, rodents, and the like. In two non-limiting examples, a subject isa human subject or a murine subject. Thus, the term “subject” includesboth human and veterinary subjects.

Therapeutic agent: Used in a generic sense, it includes treating agents,prophylactic agents, and replacement agents. A therapeutic agent is usedto ameliorate a specific set of conditions in a subject with a diseaseor a disorder.

Therapeutically effective amount: The amount of an agent (such as amatrilin-3 specific antibody or antigen binding fragment, conjugatethereof, or nucleic acid molecule encoding such molecules) that alone,or together with one or more additional agents, induces the desiredresponse, such as treatment of a cartilage disorder in a subject.Ideally, a therapeutically effective amount provides a therapeuticeffect without causing a substantial cytotoxic effect in the subject. Insome embodiments, a desired response is to increase the size or volumeof cartilage (such as growth plate cartilage or articulate cartilage),to increase the number or amount or production of chondrocytes incartilage tissue (such as growth plate or articular cartilage). Forexample, the agent or agents can increase the size or volume ofcartilage (such as growth plate cartilage or articulate cartilage), orthe number or amount or production of chondrocytes in cartilage tissue(such as growth plate or articular cartilage) by a desired amount, forexample by at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 50%, at least 75%, at least 90%, or atleast 95% as compared to a response in the absence of the agent. Inadditional examples, the agent or agents can increase the linear growthof a subject by a desired amount, for example by at least 5%, at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least50%, at least 75%, at least 90%, or at least 95% as compared to aresponse in the absence of the agent.

In some embodiments, a therapeutically effective amount of a conjugateincluding a matrilin-3 specific antibody or antigen binding fragmentlinked to a chondrogenic agent induces proliferation and differentiationof chondrocytes, for example to produce an increase in the size orvolume of growth plate cartilage tissue in a subject.

Several preparations disclosed herein are administered intherapeutically effective amounts. A therapeutically effective amount ofan antibody that specifically binds matrilin-3 or matrilin-3 bindingfragment thereof, or conjugate thereof (or a composition including oneor more of these molecules) that is administered to a human orveterinary subject will vary depending upon a number of factorsassociated with that subject, for example the overall health of thesubject. A therapeutically effective amount can be determined by varyingthe dosage and measuring the resulting therapeutic response, such as,for example, an increase in production of chondrocytes or an increase inlinear growth of the subject. Therapeutically effective amounts also canbe determined through various in vitro, in vivo or in situ immunoassays.The disclosed agents can be administered in a single dose, or in severaldoses, as needed to obtain the desired response. However, thetherapeutically effective amount of can be dependent on the sourceapplied, the subject being treated, the severity and type of thecondition being treated, and the manner of administration.

Transformed: A transformed cell is a cell into which a nucleic acidmolecule has been introduced by molecular biology techniques. As usedherein, the term transformation encompasses all techniques by which anucleic acid molecule might be introduced into such a cell, includingtransfection with viral vectors, transformation with plasmid vectors,and introduction of DNA by electroporation, lipofection, and particlegun acceleration.

Treating or preventing a disease or condition: Inhibiting the fulldevelopment of a disease or condition, for example, in a subject who isat risk of or has a disease such as a cartilage disorder and/orarthritis. “Treatment” refers to a therapeutic intervention (forexample, administration of a therapeutically effective amount of aconjugate including an antibody or antigen binding fragment thatspecifically binds matrilin-3 linked to a chondrogenic agent) thatameliorates a sign or symptom of a disease or pathological conditionafter it has begun to develop. The term “ameliorating,” with referenceto a disease or pathological condition, refers to any observablebeneficial effect of the treatment. The beneficial effect can beevidenced, for example, by a delayed onset of clinical symptoms of thedisease in a susceptible subject, a reduction in severity of some or allclinical symptoms of the disease, a slower progression of the disease,an improvement in the overall health or well-being of the subject, or byother parameters well known in the art that are specific to theparticular disease. A “prophylactic” treatment is a treatmentadministered to a subject who does not exhibit signs of a disease orexhibits only early signs for the purpose of decreasing the risk ofdeveloping pathology. In particular examples, treatment includespreventing a cartilage disorder, for example by inhibiting the fulldevelopment of a cartilage disorder. Prevention does not require a totalabsence of the cartilage disorder.

Reducing a sign or symptom of a disease or pathological conditionrelated to a disease, refers to any observable beneficial effect of thetreatment. Reducing a sign or symptom associated with a cartilagedisorder can be evidenced, for example, by a delayed onset of clinicalsymptoms of the disease in a susceptible subject, a reduction inseverity of some or all clinical symptoms of the disease (such as anincrease in linear growth of the subject), a slower progression of thedisease (for example by prolonging the life of a subject havingcartilage disorder), an improvement in the overall health or well-beingof the subject, or by other parameters well known in the art that arespecific to the particular cartilage disorder. Reducing a sign orsymptom associated with arthritis can be evidenced, for example, by adelayed onset of clinical symptoms of the disease in a susceptiblesubject, a reduction in severity of some or all clinical symptoms of thedisease (such as a decrease in pain or swelling due to the condition inthe subject), a slower progression of the disease, an improvement in theoverall health or well-being of the subject, or by other parameters wellknown in the art that are specific to the particular type of arthritis.A “prophylactic” treatment is a treatment administered to a subject whodoes not exhibit signs of a disease or exhibits only early signs for thepurpose of decreasing the risk of developing pathology.

Thus, a therapeutic agent as disclosed herein (such as a conjugatecomprising a matrilin-3 specific antibody or antigen binding fragmentlinked to a chondrogenic agent such as IGF-1) that reduces or prevents acartilage disorder and/or arthritis, can, but does not necessarilycompletely, eliminate such disorder, so long as the disorder or itssymptoms are measurably diminished, for example, by at least about 50%,such as by at least about 70%, or about 80%, or even by about 90% theinfection in the absence of the agent, or in comparison to a referenceagent.

Under conditions sufficient for: A phrase that is used to describe anyenvironment that permits a desired activity. In one example the desiredactivity is formation of an immune complex. In particular examples thedesired activity is treatment of a growth disorder.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. Recombinant DNA vectors are vectorshaving recombinant DNA. A vector can include nucleic acid sequences thatpermit it to replicate in a host cell, such as an origin of replication.A vector can also include one or more selectable marker genes and othergenetic elements known in the art. Viral vectors are recombinant nucleicacid vectors having at least some nucleic acid sequences derived fromone or more viruses. A replication deficient viral vector is a vectorthat requires complementation of one or more regions of the viral genomerequired for replication due to a deficiency in at least onereplication-essential gene function. For example, such that the viralvector does not replicate in typical host cells, especially those in ahuman patient that could be infected by the viral vector in the courseof a therapeutic method.

II. Description of Several Embodiments A. Antibodies and Antigen BindingFragments

Isolated monoclonal antibodies and antigen binding fragments thereofthat specifically bind an epitope on matrilin-3 are provided. Theantibodies can be fully human. In several embodiments the antibodies andantigen binding fragments specifically bind to matrilin-3 in thecartilage extracellular matrix. In additional embodiments, theantibodies and antigen binding fragments specifically bind to matrilin-3in the extracellular matrix of growth-plate cartilage.

In some embodiments, the antibodies and antigen binding fragmentsinclude a variable heavy (V_(H)) and a variable light (V_(L)) chain andspecifically bind matrilin-3. In several embodiments, the antibodies andantigen binding fragments include a heavy chain comprising a heavy chaincomplementarity determining region (HCDR)₁, a HCDR2 and an HCDR3, and alight chain comprising a light chain complementarity determining region(LCDR) 1, a LCDR2, and a LCDR3 and specifically bind to matrilin-3. Inseveral embodiments, the antibody or antigen binding fragment includesheavy and light chain variable regions including the HCDR1, HCDR2, andHCDR3, and LCDR1, LCDR2, and LCDR3, respectively, of one of thematrilin-3 specific clone 13, 22, or 26 antibodies, and specificallybind to matrilin-3.

The discussion of monoclonal antibodies below refers to isolatedmonoclonal antibodies and antigen binding fragments that include heavyand light chain variable domains including at least one complementaritydetermining region (CDR), such as a CDR1, CDR2 and CDR3. The person ofordinary skill in the art will understand that various CDR numberingschemes (such as the Kabat, Chothia or IMGT numbering schemes) can beused to determine CDR positions. The amino acid sequence and the CDRpositions of the heavy and light chain of the matrilin-3 antibodies(such as the clone 13, 22, and 26 antibodies) according to the IMGTnumbering scheme is shown in Table 1 (IMGT). The person of skill in theart will readily understand use of various CDR numbering schemes (suchas the Kabat numbering scheme) when referencing particular amino acidsof the antibodies disclosed herein.

TABLE 1 IMGT CDR sequences of matrilin-3 specific antibodies Clone 13SEQ ID NO: 1 A.A. Sequence SEQ ID NO: 2 A.A. Sequence HCDR1 26-33GGTFSSYA LCDR1 27-32 QDISNY HCDR2 51-58 IIPIFGTA LCDR2 50-52 DAS HCDR397-106 ARGQGYWFDP LCDR3 89-97 QQYDNLPLT Clone 22 SEQ ID NO: 3A.A. Sequence SEQ ID NO: 4 A.A. Sequence HCDR1  26-33 GDSVSSNS LCDR126-31 SLRSYY HCDR2  53-61 TYYGSKWYN LCDR2 49-51 DRD HCDR3 100-109TRGIWNAFDI LCDR3 88-97 QSYDTSLSWV  Clone 26 SEQ ID NO: 5 A.A. SequenceSEQ ID NO: 6 A.A. Sequence HCDR1 26-33 GGTFSSYA LCDR1 26-33 SSNIGSNAHCDR2 51-58 IIPILGIA LCDR2 51-53 SNN HCDR3 97-108 ARWGSGSHAFDI LCDR390-100 AAWDDSLNG WV

In some embodiments, the antibody or antigen binding fragment includesIMGT CDRs, such as those listed in Table 1. For example, in someembodiments, the antibody or antigen binding fragment includes a heavychain variable region including a HCDR1, HCDR2, and/or HCDR3 includingamino acids amino acids 26-33, 51-58, and 97-106 of SEQ ID NO: 1,respectively. In further embodiments, the antibody or antigen bindingfragment includes a heavy chain variable region including a HCDR1,HCDR2, and/or HCDR3 including amino acids amino acids 26-33, 53-61, and100-109 of SEQ ID NO: 3, respectively. In additional embodiments, theantibody or antigen binding fragment includes a heavy chain variableregion including a HCDR1, HCDR2, and/or HCDR3 including amino acids26-33, 51-58, 97-108 of SEQ ID NO: 5, respectively.

In some embodiments, the antibody or antigen binding fragment includes alight chain variable region including a LCDR1, LCDR2, and/or LCDR3including amino acids 27-32, 50-52, and 89-97 of SEQ ID NO: 2,respectively. In further embodiments, the antibody or antigen bindingfragment includes a light chain variable region including a LCDR1,LCDR2, and/or LCDR3 including amino acids 26-31, 49-51, and 88-97 of SEQID NO: 4, respectively. In additional embodiments, the antibody orantigen binding fragment includes a light chain variable regionincluding a LCDR1, LCDR2, and/or LCDR3 including amino acids 26-33,51-53, and 90-100 of SEQ ID NO: 6, respectively.

In some embodiments, the antibody or antigen binding fragment includes aheavy chain variable region including a HCDR1, HCDR2, and HCDR3including amino acids 26-33, 51-58, and 97-106 of SEQ ID NO: 1,respectively, and a light chain variable region including a LCDR1,LCDR2, and LCDR3 including amino acids 27-32, 50-52, and 89-97 of SEQ IDNO: 2, respectively. In further embodiments, the antibody or antigenbinding fragment includes a heavy chain variable region including aHCDR1, HCDR2, and HCDR3 including amino acids 26-33, 53-61, and 100-109of SEQ ID NO: 3, respectively, and a light chain variable regionincluding a LCDR1, LCDR2, and LCDR3 including amino acids 26-31, 49-51,and 88-97 of SEQ ID NO: 4, respectively. In additional embodiments, theantibody or antigen binding fragment includes a heavy chain variableregion including a HCDR1, HCDR2, and HCDR3 including amino acids 26-33,51-58, 97-108 of SEQ ID NO: 5, respectively, and a light chain variableregion including a LCDR1, LCDR2, and LCDR3 including amino acids 26-33,51-53, and 90-100 of SEQ ID NO: 6, respectively.

In some embodiments, the antibody or antigen binding fragment includes aheavy chain variable region including an amino acid sequence at least80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acidsequence set forth as one of SEQ ID NO: 1, 3, or 5. In some embodiments,the antibody or antigen binding fragment further includes at least oneamino acid substation compared to SEQ ID NO: 1, 3, or 5. In moreembodiments, the antibody or antigen binding fragment includes a lightchain variable region including an amino acid sequence at least 80%,90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence setforth as one of SEQ ID NO: 2, 4, or 6. In some embodiments, the antibodyor antigen binding fragment further includes at least one amino acidsubstation compared to SEQ ID NO: 1, 3, or 5.

In additional embodiments, the antibody or antigen binding fragmentincludes a heavy chain variable region including the amino acid sequenceset forth as one of SEQ ID NO: 1, 3, or 5. In more embodiments, theantibody or antigen binding fragment includes a light chain variableregion including the amino acid sequence set forth as one of SEQ ID NO:2, 4, or 6.

In additional embodiments, the antibody includes a heavy chain variableregion including a HCDR1, a HCDR2, and a HCDR3 including amino acidsequences at least 80% (such as at least 90%, 95%, 96%, 97%, 98%, or99%) identical to amino acids 26-33, 51-58, and/or 97-106, respectively,of SEQ ID NO: 1, and a light chain variable region including a LCDR1, aLCDR2, and a LCDR3 including amino acid sequences at least 80% (such asat least 90%, 95%, 96%, 97%, 98%, or 99%) identical to amino acids aminoacids 27-32, 50-52, and/or 89-97, respectively, of SEQ ID NO: 2. In somesuch embodiments, the antibody or antigen binding fragment furtherincludes at least one amino acid substation compared to SEQ ID NO: 1 orSEQ ID NO: 2. In additional embodiments, the antibody includes a heavychain variable region including a HCDR1, a HCDR2, and a HCDR3 includingamino acid sequences at least 80% (such as at least 90%, 95%, 96%, 97%,98%, or 99%) identical to amino acids 26-33, 53-61, and/or 100-109,respectively, of SEQ ID NO: 3, and a light chain variable regionincluding a LCDR1, a LCDR2, and a LCDR3 including amino acid sequencesat least 80% (such as at least 90%, 95%, 96%, 97%, 98%, or 99%)identical to amino acids amino acids 26-31, 49-51, and/or 88-97,respectively, of SEQ ID NO: 4. In some such embodiments, the antibody orantigen binding fragment further includes at least one amino acidsubstation compared to SEQ ID NO: 3 or SEQ ID NO: 4. In additionalembodiments, the antibody includes a heavy chain variable regionincluding a HCDR1, a HCDR2, and a HCDR3 including amino acid sequencesat least 80% (such as at least 90%, 95%, 96%, 97%, 98%, or 99%)identical to amino acids 26-33, 51-58, and/or 97-108, respectively, ofSEQ ID NO: 5, and a light chain variable region including a LCDR1, aLCDR2, and a LCDR3 including amino acid sequences at least 80% (such asat least 90%, 95%, 96%, 97%, 98%, or 99%) identical to amino acids aminoacids 26-33, 51-53, and/or 90-100, respectively, of SEQ ID NO: 6. Insome such embodiments, the antibody or antigen binding fragment furtherincludes at least one amino acid substation compared to SEQ ID NO: 5 orSEQ ID NO: 6.

In additional embodiments, the antibody or antigen binding fragmentincludes a heavy chain variable region including an amino acid sequenceat least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the aminoacid sequence set forth as SEQ ID NO: 1, and a light chain variableregion including an amino acid sequence at least 80%, 90%, 95%, 96%,97%, 98%, or 99% identical to the amino acid sequence set forth as SEQID NO: 2. In additional embodiments, the antibody or antigen bindingfragment includes a heavy chain variable region including an amino acidsequence at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to theamino acid sequence set forth as SEQ ID NO: 3, and a light chainvariable region including an amino acid sequence at least 80%, 90%, 95%,96%, 97%, 98%, or 99% identical to the amino acid sequence set forth asSEQ ID NO: 4. In additional embodiments, the antibody or antigen bindingfragment includes a heavy chain variable region including an amino acidsequence at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to theamino acid sequence set forth as SEQ ID NO: 5, and a light chainvariable region including an amino acid sequence at least 80%, 90%, 95%,96%, 97%, 98%, or 99% identical to the amino acid sequence set forth asSEQ ID NO: 6.

In some embodiments, the antibody or antigen binding fragment includes aheavy chain variable region and a light chain variable region includingthe amino acid sequences set forth as SEQ ID NOs: 1 and 2, respectively.In some embodiments, the antibody or antigen binding fragment includes aheavy chain variable region and a light chain variable region includingthe amino acid sequences set forth as SEQ ID NOs: 3 and 4, respectively.In some embodiments, the antibody or antigen binding fragment includes aheavy chain variable region and a light chain variable region includingthe amino acid sequences set forth as SEQ ID NOs: 5 and 6, respectively.

In several embodiments, the antibody or antigen binding fragment canspecifically bind matrilin-3 with an affinity of at least about1.0×10⁻⁸M, at least about 5.0×10⁻⁸M, at least about 1.0×10⁻⁹M, at leastabout 5.0×10⁻⁹M, at least about 1.0×10⁻¹° M, at least about 5.0×10⁻¹⁰ M,or at least about 1.0×10⁻¹¹ M.

The antibody or antigen binding fragment can be a human antibody orfragment thereof. Chimeric antibodies are also provided. The antibodiesand fragments can include any suitable framework region, such as (butnot limited to) a human framework region. Human framework regions, andmutations that can be made in human framework regions, are known in theart (see, for example, in U.S. Pat. No. 5,585,089, which is incorporatedherein by reference). Alternatively, a heterologous framework region,such as, but not limited to a mouse framework region, can be included inthe heavy or light chain of the antibodies. (See, for example, Jones etal., Nature 321:522, 1986; Riechmann et al., Nature 332:323, 1988;Verhoeyen et al., Science 239:1534, 1988; Carter et al., Proc. Natl.Acad. Sci. U.S.A. 89:4285, 1992; Sandhu, Crit. Rev. Biotech. 12:437,1992; and Singer et al., J. Immunol. 150:2844, 1993.)

In some embodiments, the antibody or antigen binding fragment caninclude from up to 10 amino acid substitutions (such as up to 1, 2, 3,4, 5, 6, 7, 8, or up to 9 amino acid substitutions) in the frameworkregions of the heavy chain of the antibody, or the light chain of theantibody, or the heavy and light chains of the antibody, compared to aknown framework region, or compared to the framework regions of theclone 13, 22, or 26 antibodies, and maintain specific binding activityfor matrilin-3.

In several embodiments, the antibody or antigen binding fragmentincludes at least one amino acid substitution (such as in one or more ofthe framework regions or the CDRs) compared to a naturally occurringantibody. In some embodiments, the antibody or antigen binding fragmentincludes at least one amino acid substitution (such as in one or more ofthe framework regions or the CDRs) compared to one of the clone 13, 22,or 26 antibodies as described herein.

The antibody or antigen binding fragment can be derivatized or linked toanother molecule (such as another peptide or protein). In general, theantibody or antigen binding fragment is derivatized such that thebinding to matrilin-3 is not affected adversely by the derivatization orlabeling. For example, the antibody or antigen binding fragment can befunctionally linked (by chemical coupling, genetic fusion, noncovalentassociation or otherwise) to one or more other molecular entities, suchas another antibody (for example, a bi-specific antibody or a diabody),a detectable marker, an effector molecule, or a protein or peptide thatcan mediate association of the antibody or antibody portion with anothermolecule (such as a streptavidin core region or a polyhistidine tag).

One type of derivatized antibody is produced by crosslinking two or moreantibodies (of the same type or of different types, such as to createbispecific antibodies). Suitable crosslinkers include those that areheterobifunctional, having two distinctly reactive groups separated byan appropriate spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (such as disuccinimidyl suberate). Suchlinkers are available from Pierce Chemical Company, Rockford, Ill.

The monoclonal antibodies disclosed herein can be of any isotype. Themonoclonal antibody can be, for example, an IgM or an IgG antibody, suchas IgG1, IgG2, IgG3, or an IgG4. However, in other embodiments, thedisclosed monoclonal antibodies are not an IgG. The class of an antibodythat specifically binds matrilin-3 can be switched with another (forexample, IgG can be switched to IgM), according to well-knownprocedures. For example, a nucleic acid molecule encoding the V_(L) orV_(H) of a disclosed antibody can be operatively linked to a nucleicacid sequence encoding a C_(L) or C_(H) from a different class ofimmunoglobulin molecule. This can be achieved using a vector or nucleicacid molecule that comprises a C_(L) or C_(H) chain, as known in theart. For example, an antibody that specifically binds matrilin-3, thatwas originally IgG may be class switched to an IgM. Class switching canbe used to convert one IgG subclass to another, such as from IgG1 toIgG2, IgG3, or IgG4.

In some examples, the disclosed antibodies or antigen binding fragmentsare oligomers, such as dimers, trimers, tetramers, pentamers, hexamers,septamers, octomers and so on. In some examples, the antibodies orfragments are pentamers.

In several embodiments, the constant region of the antibody includes oneor more amino acid substitutions to optimize in vivo half-life of theantibody. The serum half-life of IgG Abs can be regulated by theneonatal Fc receptor (FcRn). Thus, in several embodiments, the antibodyincludes an amino acid substitution that increases binding to the FcRn.Several such substitutions are known to the person of ordinary skill inthe art, such as substitutions at IgG constant regions T250Q and M428L(see, e.g., Hinton et al., J Immunol., 176:346-356, 2006); M428L andN434S (see, e.g., Zalevsky, et al., Nature Biotechnology, 28:157-159,2010); N434A (see, e.g., Petkova et al., Int. Immunol., 18:1759-1769,2006); T307A, E380A, and N434A (see, e.g., Petkova et al., Int.Immunol., 18:1759-1769, 2006); and M252Y, S254T, and T256E (see, e.g.,Dall'Acqua et al., J. Biol. Chem., 281:23514-23524, 2006).

In some embodiments, the constant region of the antibody includes one ofmore amino acid substitutions to reduce Antibody-dependent cell-mediatedcytotoxicity (ADCC). ADCC is mediated primarily through a set of closelyrelated Fcγ receptors. In other embodiments, the antibody include one ormore amino acid substitutions to decrease binding to FcγRIIIa.

Combinations of the above substitutions are also included, to generatean IgG constant region with increased binding to FcRn and decreasedbinding to FcγRIIIa. The combinations can, for example, increaseantibody half-life and decrease ADCC.

Antigen binding fragments of the antibodies that specifically bind tomatrilin-3 are also encompassed by the present disclosure, such assingle-domain antibodies (for example, V_(H) domain antibodies), Fab,F(ab′)₂, scFv, and Fv. These antigen binding fragments retain theability to specifically bind matrilin-3. These fragments include:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule, can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, a genetically engineered fragment containing the variable regionof the light chain and the variable region of the heavy chain expressedas two chains;

(5) Single chain antibody (such as scFv), a genetically engineeredmolecule containing the variable region of the light chain, the variableregion of the heavy chain, linked by a suitable polypeptide linker as agenetically fused single chain molecule;

(6) A dimer of a single chain antibody (scFV2), defined as a dimer of ascFV (also known as a “mini-antibody”); and

(7) V_(H) single-domain antibody, an antigen binding fragment consistingof the heavy chain variable domain.

Methods of making these fragments are known in the art (see for example,Harlow and Lane, Antibodies: A Laboratory Manual, 2nd, Cold SpringHarbor Laboratory, New York, 2013).

In some embodiments, the antigen binding fragments are Fv antibodies,which are typically about 25 kDa and contain a complete antigen-bindingsite with three CDRs per each heavy chain and each light chain. Toproduce these antibodies, the V_(H) and the V_(L) can be expressed fromtwo individual nucleic acid constructs in a host cell. If the V_(H) andthe V_(L) are expressed non-contiguously, the chains of the Fv antibodyare typically held together by noncovalent interactions. However, thesechains tend to dissociate upon dilution, so methods have been developedto crosslink the chains through glutaraldehyde, intermoleculardisulfides, or a peptide linker. Thus, in one example, the Fv can be adisulfide stabilized Fv (dsFv), wherein the heavy chain variable regionand the light chain variable region are chemically linked by disulfidebonds.

In an additional example, the Fv fragments include V_(H) and V_(L)chains connected by a peptide linker. These single-chain antigen bindingproteins (scFv) can be prepared by constructing a structural geneincluding DNA sequences encoding the V_(H) and V_(L) domains connectedby an oligonucleotide. In some embodiments, the scFv includes the aminoacid sequence set forth as one of SEQ ID NOs: 10-12. The structural genecan be inserted into an expression vector, which is subsequentlyintroduced into a host cell such as E. coli. The recombinant host cellssynthesize a single polypeptide chain with a linker peptide bridging thetwo V domains. Methods for producing scFvs are known in the art (seeWhitlow et al., Methods: a Companion to Methods in Enzymology, Vol. 2,page 97, 1991; Bird et al., Science 242:423, 1988; U.S. Pat. No.4,946,778; Pack et al., Bio/Technology 11:1271, 1993; Ahmad et al.,Clin. Dev. Immunol., 2012, doi:10.1155/2012/980250; Marbry, IDrugs,13:543-549, 2010). Dimers of a single chain antibody (scFV₂), are alsocontemplated.

Antigen binding fragments can be prepared by proteolytic hydrolysis ofthe antibody or by expression in a host cell (such as an E. coli cell)of DNA encoding the fragment. Antigen binding fragments can also beobtained by pepsin or papain digestion of whole antibodies byconventional methods. For example, antigen binding fragments can beproduced by enzymatic cleavage of antibodies with pepsin to provide a 5Sfragment denoted F(ab′)₂. This fragment can be further cleaved using athiol reducing agent, and optionally a blocking group for the sulfhydrylgroups resulting from cleavage of disulfide linkages, to produce 3.5SFab′ monovalent fragments. Alternatively, an enzymatic cleavage usingpepsin produces two monovalent Fab′ fragments and an Fc fragmentdirectly (see U.S. Pat. Nos. 4,036,945 and 4,331,647, and referencescontained therein; Nisonhoff et al., Arch. Biochem. Biophys. 89:230,1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., Methods inEnzymology, Vol. 1, page 422, Academic Press, 1967; and Coligan et al.at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

One of skill will realize that conservative variants of the antibodiescan be produced. Such conservative variants employed in antigen bindingfragments, such as dsFv fragments or in scFv fragments, will retaincritical amino acid residues necessary for correct folding andstabilizing between the V_(H) and the V_(L) regions, and will retain thecharge characteristics of the residues in order to preserve the low pIand low toxicity of the molecules Amino acid substitutions (such as atmost one, at most two, at most three, at most four, or at most fiveamino acid substitutions) can be made in the V_(H) or the V_(L) regionsto increase yield. In particular examples, the V_(H) sequence is one ofSEQ ID NO: 1, 3, or 5. In other examples, the V_(L) sequence is one ofSEQ ID NO: 2, 4, or 6. Conservative amino acid substitution tablesproviding functionally similar amino acids are well known to one ofordinary skill in the art.

Also included are antibodies that bind to the same epitope on matrilin-3to which the matrilin-3 specific antibodies provided herein (e.g., clone13, 22, or 26) bind. Antibodies that bind to such an epitope can beidentified based on their ability to cross-compete (for example, tocompetitively inhibit the binding of, in a statistically significantmanner) with the matrilin-3 specific antibodies provided herein inmatrilin-3 binding assays (such as those described in the Examples). Anantibody “competes” for binding when the competing antibody inhibitsmatrilin-3 binding of an antibody of the invention by more than 50%, inthe presence of competing antibody concentrations higher than 10⁶×K_(D)of the competing antibody. In a certain embodiment, the antibody thatbinds to the same epitope on matrilin-3 as the antibodies of the presentinvention is a human monoclonal antibody. Such human monoclonalantibodies can be prepared and isolated as described herein.

Additionally, to increase binding affinity of the antibody, the V_(L)and V_(H) segments can be randomly mutated, such as within H-CDR3 regionor the L-CDR3 region, in a process analogous to the in vivo somaticmutation process responsible for affinity maturation of antibodiesduring a natural immune response. Thus in vitro affinity maturation canbe accomplished by amplifying V_(H) and V_(L) regions using PCR primerscomplementary to the H-CDR3 or L-CDR3, respectively. In this process,the primers have been “spiked” with a random mixture of the fournucleotide bases at certain positions such that the resultant PCRproducts encode V_(H) and V_(L) segments into which random mutationshave been introduced into the V_(H) and/or V_(L) CDR3 regions. Theserandomly mutated V_(H) and V_(L) segments can be tested to determine thebinding affinity for matrilin-3. In particular examples, the V_(H) aminoacid sequence is one of SEQ ID NOs: 1, 3, or 5. In other examples, theV_(L) amino acid sequence is SEQ ID NOs: 2, 4, or 6.

B. Conjugates

Monoclonal antibodies specific for matrilin-3, or antigen bindingfragments thereof, can be conjugated to an agent, such as an effectormolecule or detectable marker, using any number of means known to thoseof skill in the art. The effector molecule may be heterologous and/ornon-naturally occurring. Both covalent and noncovalent attachment meansmay be used. One of skill in the art will appreciate that variouseffector molecules and detectable markers can be used, including (butnot limited to) chondrogenic agents, anti-arthritis agents, andradioactive agents such as ¹²⁵I, ³²P, ¹⁴C, ³H and ³⁵S and other labels,target moieties and ligands, etc. In several embodiments, the effectormolecule is a polypeptide and is linked (for example by a heterologouspeptide linker) to the N-terminus of an scFv including the heavy andlight chain CDRs of a matrilin-3 specific antibody, such as the clone13, clone 22, or clone 26 antibody.

The choice of a particular effector molecule or detectable markerdepends on the particular target molecule or cell, and the desiredbiological effect. Thus, for example, the effector molecule can be achondrogenic agent that is used to promote or stimulate chondrogenesisin cartilage tissue.

The procedure for attaching an effector molecule or detectable marker toan antibody or antigen binding fragment varies according to the chemicalstructure of the effector. Polypeptides typically contain a variety offunctional groups; such as carboxylic acid (COOH), free amine (—NH₂) orsulfhydryl (—SH) groups, which are available for reaction with asuitable functional group on an a polypeptide to result in the bindingof the effector molecule or detectable marker. Alternatively, theantibody or antigen binding fragment is derivatized to expose or attachadditional reactive functional groups. The derivatization may involveattachment of any of a number of known linker molecules such as thoseavailable from Pierce Chemical Company, Rockford, IL. The linker can beany molecule used to join the antibody or antigen binding fragment tothe effector molecule or detectable marker. The linker is capable offorming covalent bonds to both the antibody or antigen binding fragmentand to the effector molecule or detectable marker. Suitable linkers arewell known to those of skill in the art and include, but are not limitedto, straight or branched-chain carbon linkers, heterocyclic carbonlinkers, or peptide linkers. Where the antibody or antigen bindingfragment and the effector molecule or detectable marker arepolypeptides, the linkers may be joined to the constituent amino acidsthrough their side groups (such as through a disulfide linkage tocysteine) or to the alpha carbon amino and carboxyl groups of theterminal amino acids.

In several embodiments, the linker can include a spacer element, which,when present, increases the size of the linker such that the distancebetween the effector molecule or the detectable marker and the antibodyor antigen binding fragment is increased. Exemplary spacers are known tothe person of ordinary skill, and include those listed in U.S. Pat. Nos.7,964,5667, 498,298, 6,884,869, 6,323,315, 6,239,104, 6,034,065,5,780,588, 5,665,860, 5,663,149, 5,635,483, 5,599,902, 5,554,725,5,530,097, 5,521,284, 5,504,191, 5,410,024, 5,138,036, 5,076,973,4,986,988, 4,978,744, 4,879,278, 4,816,444, and 4,486,414, as well asU.S. Pat. Pub. Nos. 20110212088 and 20110070248, each of which isincorporated by reference in its entirety.

In yet other specific embodiments, the linker is a malonate linker(Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyllinker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a3′-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

In several embodiments, the linker is resistant to cleavage in anextracellular environment. For example, no more than about 20%, no morethan about 15%, no more than about 10%, no more than about 5%, no morethan about 3%, or no more than about 1% of the linkers, in a sample ofconjugate, are cleaved when the conjugate is present in an extracellularenvironment (for example, in plasma). Whether or not a linker isresistant to cleavage in an extracellular environment can be determined,for example, by incubating the conjugate containing the linker ofinterest with plasma for a predetermined time period (for example, 2, 4,8, 16, or 24 hours) and then quantitating the amount of free effectormolecule or detectable marker present in the plasma. A variety ofexemplary linkers that can be used in conjugates are described in WO2004010957, U.S. Publication No. 2006/0074008, U.S. Publication No.20050238649, and U.S. Publication No. 2006/0024317, each of which isincorporated by reference herein in its entirety.

In several embodiments, conjugates of an antibody or antigen bindingfragment and one or more chondrogenic agents, are provided. Non-limitingexamples of such chondrogenic agents include growth-regulating endocrinesignaling molecules (e.g., growth hormone, IGF-1, steroids (e.g., anestrogen, an androgen, estradiol)), growth-regulating paracrinesignaling molecules (e.g., Indian Hedgehog (IHH), bone morphogeneticproteins (BMPs), C-type natriuretic peptide (CNP), WNTs, and FGFs). Inseveral embodiments, a chondrogenic agent is conjugated to a matrilin-3specific antibody or antigen binding fragment (such as an antibody orantigen binding fragment including the CDRs of the clone 13, clone 22,or clone 26 antibodies disclosed herein).

In some embodiments the antibody or antigen binding fragment (such as anantibody or antigen binding fragment including the CDRs of one of theclone 13, clone 22, or clone 26 matrilin-3 specific antibodies) isconjugated to a growth hormone or fragment thereof that induceschondrogenesis. Human growth hormone is used for both growthhormone-deficiency and certain non-growth hormone-deficient causes ofshort stature (Richmond, Current Indications for Growth Hormone TherapyVol. 18, Karger, 2010). Exemplary human growth hormone polypeptide andnucleic acid sequences are known to the person of ordinary skill in theart. For example, the polypeptide sequence of a human growth hormoneprecursor is provided in NCBI Ref. NP_000506.2 (incorporated byreference herein as present in the database on Dec. 1, 2103)

(SEQ ID NO: 18) MATGSRTSLLLAFGLLCLPWLQEGSAFPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDM DKVETFLRIVQCRSVEGSCGF

Further, the person of ordinary skill in the art can readily determinethe polypeptide sequence of a mature growth hormone; for example maturehuman growth hormone can include a polypeptide sequence set forth asfollows:

(SEQ ID NO: 19) FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF

An exemplary nucleic acid sequence encoding a human growth hormone isset forth as NCBI Ref. NM_000515.3 (incorporated by reference herein aspresent in the database on Dec. 1, 2103)

(SEQ ID NO: 20) Aggatcccaaggcccaactccccgaaccactcagggtcctgtggacagctcacctagctgcaatggctacaggctcccggacgtccctgctcctggcttttggcctgctctgcctgccctggcttcaagagggcagtgccttcccaaccattcccttatccaggctttttgacaacgctatgctccgcgcccatcgtctgcaccagctggcctttgacacctaccaggagtttgaagaagcctatatcccaaaggaacagaagtattcattcctgcagaacccccagacctccctctgtttctcagagtctattccgacaccctccaacagggaggaaacacaacagaaatccaacctagagctgctccgcatctccctgctgctcatccagtcgtggctggagcccgtgcagttcctcaggagtgtcttcgccaacagcctggtgtacggcgcctctgacagcaacgtctatgacctcctaaaggacctagaggaaggcatccaaacgctgatggggaggctggaagatggcagcccccggactgggcagatcttcaagcagacctacagcaagttcgacacaaactcacacaacgatgacgcactactcaagaactacgggctgctctactgcttcaggaaggacatggacaaggtcgagacattcctgcgcatcgtgcagtgccgctctgtggagggcagctgtggcttctagctgcccgggtggcatccctgtgacccctccccagtgcctctcctggccctggaagttgccactccagtgcccaccagccttgtcctaataaaattaagttgcatca

In some embodiments the antibody or binding fragment (such as anantibody or antigen binding fragment including the CDRs of one of theclone 13, clone 22, or clone 26 matrilin-3 specific antibodies) isconjugated to an IGF-1 or fragment thereof that induces chondrogenesis.Exemplary IGF-1 polypeptide and nucleic acid sequences are known to theperson of ordinary skill in the art. For example, the polypeptidesequence of IGF-1 precursor is set forth as UniProt Ref. No. P05019.1,incorporated by reference herein as present in the database on Dec. 1,2103):

(SEQ ID NO: 21) MGKISSLPTQLFKCCFCDFLKVKMHTMSSSHLFYLALCLLTFTSSATAGPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSARSVRAQRHTDMPKTQKYQPPSTNKNTKSQRRKGWPKTHPGGEQKEGTEASLQIRGKKKEQRREIGSRNAECRGKKGK

Further, the person of ordinary skill in the art can readily determinethe polypeptide sequence of a mature IGF-1; for example mature IGF-1 caninclude a polypeptide sequence set forth as follows:

(SEQ ID NO: 22) GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPAKSA

In another example, the mature IGF-1 can include a polypeptide sequenceset forth as

(SEQ ID NO: 49) GPETLCGAELVDALQFVCGDRGFYFNKPTGYGSSSRRAPQTGIVDECCFRSCDLRRLEMYCAPL

An exemplary nucleic acid sequence encoding a human IGF-1 is set forthas GENBANK™ Ref. No. X00173.1, incorporated by reference herein aspresent in the database on Dec. 1, 2103)

(SEQ ID NO: 23) Cttcagaagcaatgggaaaaatcagcagtcttccaacccaattatttaagtgctgcttttgtgatttcttgaaggtgaagatgcacaccatgtcctcctcgcatctcttctacctggcgctgtgcctgctcaccttcaccagctctgccacggctggaccggagacgctctgcggggctgagctggtggatgctcttcagttcgtgtgtggagacaggggcttttatttcaacaagcccacagggtatggctccagcagtcggagggcgcctcagacaggtatcgtggatgagtgctgcttccggagctgtgatctaaggaggctggagatgtattgcgcacccctcaagcctgccaagtcagctcgctctgtccgtgcccagcgccacaccgacatgcccaagacccagaaggaagtacatttgaagaacgcaagtagagggagtgcaggaaacaagaactacaggatgtaggaagaccctcctgaggagtgaagagtgacatgccaccgcaggatcctttgctctgcacgagttacctgttaaactttggaacacctaccaaaaaataagtttgataacatttaaaagatgggcgtttcccccaatgaaatacacaagtaaacattccaacattgtctttaggagtgatttgcaccttgcaaaaatggtcctggagttggtagattgctgttgatcttttatcaataatgttctatagaaaag

In some embodiments the antibody or antigen binding fragment (such as anantibody or antigen binding fragment including the CDRs of one of theclone 13, clone 22, or clone 26 matrilin-3 specific antibodies asdisclosed herein) is conjugated to an Indian Hedgehog (IHH) protein orfragment thereof that induces chondrogenesis. IHH is known to stimulategrowth plate chondrogenesis (Chau, et al. J Mol Endocrinol 47, 99-107,2011; Kobayashi, et al. J Clin Invest 115, 1734-1742, 2005; Kronenberg.Ann N Y Acad Sci 1068, 1-13, 2006; Maeda, et al. Proc Natl Acad Sci USA104, 6382-6387, 2007; Long, et al. Dev Biol 298, 327-333, 2006; Amizuka,et al. J Cell Biol 126, 1611-1623, 1994; Long, et al. Development 128,5099-5108, 2001; Mak, et al. Development 135, 1947-1956, 2008).Exemplary IHH polypeptide and nucleic acid sequences are known to theperson of ordinary skill in the art. For example, the polypeptidesequence of an IHH precursor is set forth as NCBI Ref. No. NP_002172.2,incorporated by reference herein as present in the database on Dec. 1,2103):

(SEQ ID NO: 24) MSPARLRPRLHFCLVLLLLLVVPAAWGCGPGRVVGSRRRPPRKLVPLAYKQFSPNVPEKTLGASGRYEGKIARSSERFKELTPNYNPDIIFKDEENTGADRLMTQRCKDRLNSLAISVMNQWPGVKLRVTEGWDEDGHHSEESLHYEGRAVDITTSDRDRNKYGLLARLAVEAGFDWVYYESKAHVHCSVKSEHSAAAKTGGCFPAGAQVRLESGARVALSAVRPGDRVLAMGEDGSPTFSDVLIFLDREPHRLRAFQVIETQDPPRRLALTPAHLLFTADNHTEPAARFRATFASHVQPGQYVLVAGVPGLQPARVAAVSTHVALGAYAPLTKHGTLVVEDVVASCFAAVADHHLAQLAFWPLRLFHSLAWGSWTPGEGVHWYPQLLYRLGRLLLEEGS FHPLGMSGAGS

Further, the person of ordinary skill in the art can readily determinethe polypeptide sequence of a mature IHH; for example mature IHH caninclude a polypeptide sequence set forth as follows:

(SEQ ID NO: 25) GPGRVVGSRRRPPRKLVPLAYKQFSPNVPEKTLGASGRYEGKIARSSERFKELTPNYNPDIIFKDEENTGADRLMTQRCKDRLNSLAISVMNQWPGVKLRVTEGWDEDGHHSEESLHYEGRAVDITTSDRDRNKYGLLARLAVEAGFDWVYYESKAHVHCSVKSEHSAAAKTGGCFPAGAQVRLESGARVALSAVRPGDRVLAMGEDGSPTFSDVLIFLDREPHRLRAFQVIETQDPPRRLALTPAHLLFTADNHTEPAARFRATFASHVQPGQYVLVAGVPGLQPARVAAVSTHVALGAYAPLTKHGTLVVEDVVASCFAAVADHHLAQLAFWPLRLFHSLAWGSWTPGEGVHWYPQLLYRLGRLLLEEGSFHPLGMSGAGS

An exemplary nucleic acid sequence encoding IHH is set forth as NCBIRef. No. NM_002181.3, incorporated by reference herein as present in thedatabase on Dec. 1, 2103)

(SEQ ID NO: 26) atcagcccaccaggagacctcgcccgccgctcccccgggctccccggccatgtctcccgcccggctccggccccgactgcacttctgcctggtcctgttgctgctgctggtggtgccggcggcatggggctgcgggccgggtcgggtggtgggcagccgccggcgaccgccacgcaaactcgtgccgctcgcctacaagcagttcagccccaatgtgcccgagaagaccctgggcgccagcggacgctatgaaggcaagatcgctcgcagctccgagcgcttcaaggagctcacccccaattacaatccagacatcatcttcaaggacgaggagaacacaggcgccgaccgcctcatgacccagcgctgcaaggaccgcctgaactcgctggctatctcggtgatgaaccagtggcccggtgtgaagctgcgggtgaccgagggctgggacgaggacggccaccactcagaggagtccctgcattatgagggccgcgcggtggacatcaccacatcagaccgcgaccgcaataagtatggactgctggcgcgcttggcagtggaggccggctttgactgggtgtattacgagtcaaaggcccacgtgcattgctccgtcaagtccgagcactcggccgcagccaagacgggcggctgcttccctgccggagcccaggtacgcctggagagtggggcgcgtgtggccttgtcagccgtgaggccgggagaccgtgtgctggccatgggggaggatgggagccccaccttcagcgatgtgctcattttcctggaccgcgagcctcacaggctgagagccttccaggtcatcgagactcaggaccccccacgccgcctggcactcacacccgctcacctgctctttacggctgacaatcacacggagccggcagcccgcttccgggccacatttgccagccacgtgcagcctggccagtacgtgctggtggctggggtgccaggcctgcagcctgcccgcgtggcagctgtctctacacacgtggccctcggggcctacgccccgctcacaaagcatgggacactggtggtggaggatgtggtggcatcctgcttcgcggccgtggctgaccaccacctggctcagttggccttctggcccctgagactctttcacagcttggcatggggcagctggaccccgggggagggtgtgcattggtacccccagctgctctaccgcctggggcgtctcctgctagaagagggcagcttccacccactgggcatgtccggggcagggagctgaaaggactccaccgctgccctcctggaactgctgtactgggtccagaagcctctcagccaggagggagctggccctggaagggacctgagctgggggacactggctcctgccatctcctctgccatgaagatacaccattgagacttgactgggcaacaccagcgtcccccacccccgtcgtggtgtagtcatagagctgcaagctgagctggcgaggggatggttgttgacccctctctcctagagaccttgaggctggcacggcgactcccaactcagcctgctctcactacgagttttcatactctgcctcccccattggggagggcccattccatccatcttaggcccctttgggtgggcttgcgcctcagttgatgctgctaaattccctgggagccagcatggatctggctggacccgatgctgtccagaactgggaaggccacaggggtggggcagccatcccggccattctgaggtatgacattcctccccggccacactcctcaagacacatccagagactgttgctgtctgtgggcagagttctgtgttctggccaatgtgaccgtagtgccggggactgggggaggtgggttggatgtgcttgccacccccccggctaagctcccccttctgctgaaccatgatccccaccccctccgccggtcagtctcccataccttatttattggagtggagggggaagcccatgggagaattttggggatgttttggtcttttcttccttttgtaataaaaattatttaagttgttagagccaaa

In some embodiments the antibody or antigen binding fragment (such as anantibody or antigen binding fragment including the CDRs of one of theclone 13, clone 22, or clone 26 matrilin-3 specific antibodies) isconjugated to a bone morphogenic protein (BMP) or fragment thereof thatinduces chondrogenesis. BMPs are known to stimulate growth platechondrogenesis (De Luca, et al. Endocrinology 142, 430-436, 2001;Nilsson, et al. J Endocrinol 193, 75-84, 2007; Kobayashi, et al. ProcNatl Acad Sci USA 102, 18023-18027, 2005; Yoon, et al. Development 133,4667-4678, 2006; Wu, et al. J Biol Chem 286, 24726-24734, 2011; Yoon, etal. Proc Natl Acad Sci USA 102, 5062-5067, 2005). Exemplary BMPpolypeptide and nucleic acid sequences are known to the person ofordinary skill in the art. For example, polypeptide and encoding nucleicacid sequences of BMP1 (NM_001199.3; NP_001190.1), BMP2 (NM_001200.2;NP_001191.1), BMP3 (NM_001201.2; NP_001192.2), BMP4 (NM_001202.3;NP_001193.2), BMP6 (NM_021073.2; NP_066551.1), BMP7 (NM_001719.2;NP_001710.1), BMP8A (NM_181809.3; NP_861525.2) are known, and theindicated NCBI Ref. Nos. are each incorporated by reference herein aspresent in the database on Dec. 1, 2013. In one non-limiting example,the polypeptide sequence of a BMP1 precursor is set forth as NCBI Ref.No. NP_001190.1, incorporated by reference herein as present in thedatabase on Dec. 1, 2103):

(SEQ ID NO: 27) MPGVARLPLLLGLLLLPRPGRPLDLADYTYDLAEEDDSEPLNYKDPCKAAAFLGDIALDEEDLRAFQVQQAVDLRRHTARKSSIKAAVPGNTSTPSCQSTNGQPQRGACGRWRGRSRSRRAATSRPERVWPDGVIPFVIGGNFTGSQRAVFRQAMRHWEKHTCVTFLERTDEDSYIVFTYRPCGCCSYVGRRGGGPQAISIGKNCDKFGIVVHELGHVVGFWHEHTRPDRDRHVSIVRENIQPGQEYNFLKMEPQEVESLGETYDFDSIMHYARNTFSRGIFLDTIVPKYEVNGVKPPIGQRTRLSKGDIAQARKLYKCPACGETLQDSTGNFSSPEYPNGYSAHMHCVWRISVTPGEKIILNFTSLDLYRSRLCWYDYVEVRDGFWRKAPLRGRFCGSKLPEPIVSTDSRLWVEFRSSSNWVGKGFFAVYEAICGGDVKKDYGHIQSPNYPDDYRPSKVCIWRIQVSEGFHVGLTFQSFEIERHDSCAYDYLEVRDGHSESSTLIGRYCGYEKPDDIKSTSSRLWLKFVSDGSINKAGFAVNFFKEVDECSRPNRGGCEQRCLNTLGSYKCSCDPGYELAPDKRRCEAACGGFLTKLNGSITSPGWPKEYPPNKNCIWQLVAPTQYRISLQFDFFETEGNDVCKYDFVEVRSGLTADSKLHGKFCGSEKPEVITSQYNNMRVEFKSDNTVSKKGFKAHFFSEKRPALQPPRGRPHQLKFRVQKRNRTPQ

Further, the person of ordinary skill in the art can readily determinethe polypeptide sequence of a mature BMP; for example the polypeptidesequence of mature BMP1 is set forth as follows:

(SEQ ID NO: 28) LDLADYTYDLAEEDDSEPLNYKDPCKAAAFLGDIALDEEDLRAFQVQQAVDLRRHTARKSSIKAAVPGNTSTPSCQSTNGQPQRGACGRWRGRSRSRRAATSRPERVWPDGVIPFVIGGNFTGSQRAVFRQAMRHWEKHTCVTFLERTDEDSYIVFTYRPCGCCSYVGRRGGGPQAISIGKNCDKFGIVVHELGHVVGFWHEHTRPDRDRHVSIVRENIQPGQEYNFLKMEPQEVESLGETYDFDSIMHYARNTFSRGIFLDTIVPKYEVNGVKPPIGQRTRLSKGDIAQARKLYKCPACGETLQDSTGNFSSPEYPNGYSAHMHCVWRISVTPGEKIILNFTSLDLYRSRLCWYDYVEVRDGFWRKAPLRGRFCGSKLPEPIVSTDSRLWVEFRSSSNWVGKGFFAVYEAICGGDVKKDYGHIQSPNYPDDYRPSKVCIWRIQVSEGFHVGLIFQSFEIERHDSCAYDYLEVRDGHSESSTLIGRYCGYEKPDDIKSTSSRLWLKFVSDGSINKAGFAVNFFKEVDECSRPNRGGCEQRCLNTLGSYKCSCDPGYELAPDKRRCEAACGGFLTKLNGSITSPGWPKEYPPNKNCIWQLVAPTQYRISLQFDFFETEGNDVCKYDFVEVRSGLTADSKLHGKFCGSEKPEVITSQYNNMRVEFKSDNTVSKKGFKAHFFSEKRPALQPPRGRPHQLKFRV QKRNRTPQ

An exemplary nucleic acid sequence encoding BMP1 is set forth as NCBIRef. No. NM_001199.3, incorporated by reference herein as present in thedatabase on Dec. 1, 2103)

(SEQ ID NO: 29) gtcggagggagggagggagggagagaaagaaagagagaaaaagaaggaaagggagagggagacggctggagcccgaggacgagcgcggagccgcggaccgagcggggggcgggagacaggaaggagggaggcgagcagagggaaggggaagaggtcggggagcgagggcgggagcggtcgcggtcgcgatcgagcaagcaagcgggcgagaggacgccctcccctggcctccagtgcgccgcttccctcgccgccgccccgccagcatgcccggcgtggcccgcctgccgctgctgctcgggctgctgctgctcccgcgtcccggccggccgctggacttggccgactacacctatgacctggcggaggaggacgactcggagcccctcaactacaaagacccctgcaaggcggctgcctttcttggggacattgccctggacgaagaggacctgagggccttccaggtacagcaggctgtggatctcagacggcacacagctcgtaagtcctccatcaaagctgcagttccaggaaacacttctacccccagctgccagagcaccaacgggcagcctcagaggggagcctgtgggagatggagaggtagatcccgtagccggcgggcggcgacgtcccgaccagagcgtgtgtggcccgatggggtcatcccctttgtcattgggggaaacttcactggtagccagagggcagtcttccggcaggccatgaggcactgggagaagcacacctgtgtcaccttcctggagcgcactgacgaggacagctatattgtgttcacctatcgaccttgcgggtgctgctcctacgtgggtcgccgcggcgggggcccccaggccatctccatcggcaagaactgtgacaagttcggcattgtggtccacgagctgggccacgtcgtcggcttctggcacgaacacactcggccagaccgggaccgccacgtttccatcgttcgtgagaacatccagccagggcaggagtataacttcctgaagatggagcctcaggaggtggagtccctgggggagacctatgacttcgacagcatcatgcattacgctcggaacacattctccaggggcatcttcctggataccattgtccccaagtatgaggtgaacggggtgaaacctcccattggccaaaggacacggctcagcaagggggacattgcccaagcccgcaagctttacaagtgcccagcctgtggagagaccctgcaagacagcacaggcaacttctcctcccctgaataccccaatggctactctgctcacatgcactgcgtgtggcgcatctctgtcacacccggggagaagatcatcctgaacttcacgtccctggacctgtaccgcagccgcctgtgctggtacgactatgtggaggtccgagatggcttctggaggaaggcgcccctccgaggccgcttctgcgggtccaaactccctgagcctatcgtctccactgacagccgcctctgggttgaattccgcagcagcagcaattgggttggaaagggcttctttgcagtctacgaagccatctgcgggggtgatgtgaaaaaggactatggccacattcaatcgcccaactacccagacgattaccggcccagcaaagtctgcatctggcggatccaggtgtctgagggcttccacgtgggcctcacattccagtcctttgagattgagcgccacgacagctgtgcctacgactatctggaggtgcgcgacgggcacagtgagagcagcaccctcatcgggcgctactgtggctatgagaagcctgatgacatcaagagcacgtccagccgcctctggctcaagttcgtctctgacgggtccattaacaaagcgggctttgccgtcaactttttcaaagaggtggacgagtgctctcggcccaaccgcgggggctgtgagcagcggtgcctcaacaccctgggcagctacaagtgcagctgtgaccccgggtacgagctggccccagacaagcgccgctgtgaggctgcttgtggcggattcctcaccaagctcaacggctccatcaccagcccgggctggcccaaggagtacccccccaacaagaactgcatctggcagctggtggcccccacccagtaccgcatctccctgcagtttgacttctttgagacagagggcaatgatgtgtgcaagtacgacttcgtggaggtgcgcagtggactcacagctgactccaagctgcatggcaagttctgtggttctgagaagcccgaggtcatcacctcccagtacaacaacatgcgcgtggagttcaagtccgacaacaccgtgtccaaaaagggcttcaaggcccacttcttctcagaaaagaggccagctctgcagccccctcggggacgcccccaccagctcaaattccgagtgcagaaaagaaaccggaccccccagtgaggcctgccaggcctcccggaccccttgttactcaggaacctcaccttggacggaatgggatgggggcttcggtgcccaccaaccccccacctccactctgccattccggcccacctccctctggccggacagaactggtgctctcttctccccactgtgcccgtccgcggaccggggacccttccccgtgccctaccccctcccattttgatggtgtctgtgacatttcctgttgtgaagtaaaagagggacccctgcgtcctgctcctttctcttgcagaaaaaaaa

In some embodiments the antibody or antigen binding fragment (such as anantibody or antigen binding fragment including the CDRs of one of theclone 13, clone 22, or clone 26 matrilin-3 specific antibodies) isconjugated to a CNP or fragment thereof that induces chondrogenesis. CNPvariants can also be including in the disclosed conjugates; several CNPvariants are known, including those described in U.S. Pat. App. Pub. No.20130096061, 20120316114, and 2004/0138134, each of which isincorporated by reference herein in its entirety. CNPs are known topromote growth plate chondrogenesis (Mericq, et al. Pediatr Res 47,189-193, 2000; Agoston, et al. BMC Dev Biol 7, 18, 2007; Olney, et al. JClin Endocrinol Metab 92, 4294-4298, 2007; Olney, et al. J ClinEndocrinol Metab 91, 1229-1232, 2006; Teixeira, et al. Dev Biol 319,171-178, (2008); Woods, et al. Endocrinology 148, 5030-5041, 2007).Exemplary CNP polypeptide and nucleic acid sequences are known to theperson of ordinary skill in the art. In one non-limiting example, apolypeptide sequence of a CNP precursor is set forth as NCBI Ref. No.NP_077720.1, incorporated by reference herein as present in the databaseon Dec. 1, 2103):

(SEQ ID NO: 30) MHLSQLLACALLLTLLSLRPSEAKPGAPPKVPRTPPAEELAEPQAAGGGQKKGDKAPGGGGANLKGDRSRLLRDLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC

Several active fragments of CNP are known to the person of ordinaryskill in the art, including CNP-53(DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 31),CNP-29 (YKGANKKGLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 32), and CNP-22(GLSKGCFGLKLDRIGSMSGLGC; SEQ ID NO: 33). These active fragments of CNPcan be linked to the antibody or antigen binding fragment.

An exemplary nucleic acid sequence encoding BMP1 is set forth as NCBIRef. No. NM_024409.2, incorporated by reference herein as present in thedatabase on Dec. 1, 2103)

(SEQ ID NO: 34) Cgcatccccctgctggtctgcccgccgacctgcgcgccctcgctgccgcccgtgtgcgcccctcgaccccagcggcaccatgcatctctcccagctgctggcctgcgccctgctgctcacgctgctctccctccggccctccgaagccaagcccggggcgccgccgaaggtcccgcgaaccccgccggcagaggagctggccgagccgcaggctgcgggcggcggtcagaagaagggcgacaaggctcccgggggcgggggcgccaatctcaagggcgaccggtcgcgactgctccgggacctgcgcgtggacaccaagtcgcgggcagcgtgggctcgccttctgcaagagcaccccaacgcgcgcaaatacaaaggagccaacaagaagggcttgtccaagggctgcttcggcctcaagctggaccgaatcggctccatgagcggcctgggatgttagtgcggcgccccctggcggcggatcgggaactggctccgttgtgctgaggtcatctttggtcatcagcctccagcatctggaaacacctccaacgcaatgtggcttttacatttctttctttctttctttttttttcctggtactgggaatacacaacaccagctgttttattattatttggggagggggttgtgattttattatttgtttttttaaaatgaaaaataaaaagttatatat t

In some embodiments the antibody or antigen binding fragment (such as anantibody or antigen binding fragment including the CDRs of one of theclone 13, clone 22, or clone 26 matrilin-3 specific antibodies) isconjugated to a Wnt protein or fragment thereof that induceschondrogenesis. WNT proteins are known to promote chondrogenesis (see,e.g., Andrade, et al. Bone 40, 1361-1369, 2007; Hartmann, et al.Development 127, 3141-3159, 2000; Yates, et al. DNA Cell Biol 24,446-457, 2005; Yang, et al. Development 130, 1003-1015, 2003; Akiyama,et al., Genes Dev 18, 1072-1087, 2004). Exemplary Wnt polypeptide andnucleic acid sequences are known to the person of ordinary skill in theart. For example, polypeptide and encoding nucleic acid sequences ofWnt1 (NP_005421.1; NM_005430.3), Wnt2 (NM_003391.2; NP_003382.1), Wnt3(NM_030753.4; NP_110380.1), Wnt4 (NM_030761.4; NP_110388.2), Wnt5(NM_001256105.1; NP_001243034.1), are known, each of which isincorporated by reference herein as present in the database on Dec. 1,2013. In one non-limiting example, the polypeptide sequence of Wnt1precursor is set forth as NCBI Ref. No. NP_005421.1, incorporated byreference herein as present in the database on Dec. 1, 2103)

(SEQ ID NO: 35) MGLWALLPGWVSATLLLALAALPAALAANSSGRWWGIVNVASSTNLLTDSKSLQLVLEPSLQLLSRKQRRLIRQNPGILHSVSGGLQSAVRECKWQFRNRRWNCPTAPGPHLFGKIVNRGCRETAFIFAITSAGVTHSVARSCSEGSIESCTCDYRRRGPGGPDWHWGGCSDNIDFGRLFGREFVDSGEKGRDLRFLMNLHNNEAGRTTVFSEMRQECKCHGMSGSCTVRTCWMRLPTLRAVGDVLRDRFDGASRVLYGNRGSNRASRAELLRLEPEDPAHKPPSPHDLVYFEKSPNFCTYSGRLGTAGTAGRACNSSSPALDGCELLCCGRGHRTRTQRVTERCNCTFH WCCHVSCRNCTHTRVLHECL

Further, the person of ordinary skill in the art can readily determinethe polypeptide sequence of mature Wnts; for example the polypeptidesequence of mature Wnt1 is set forth as follows:

(SEQ ID NO: 36) ANSSGRWWGIVNVASSTNLLTDSKSLQLVLEPSLQLLSRKQRRLIRQNPGILHSVSGGLQSAVRECKWQFRNRRWNCPTAPGPHLFGKIVNRGCRETAFIFAITSAGVTHSVARSCSEGSIESCTCDYRRRGPGGPDWHWGGCSDNIDFGRLFGREFVDSGEKGRDLRFLMNLHNNEAGRTTVFSEMRQECKCHGMSGSCTVRTCWMRLPTLRAVGDVLRDRFDGASRVLYGNRGSNRASRAELLRLEPEDPAHKPPSPHDLVYFEKSPNFCTYSGRLGTAGTAGRACNSSSPALDGCELLCCGRGHRTRTQRVTERCNCTFHWCCHVSCRNCTHTRVLHECL

An exemplary nucleic acid sequence encoding Wnt1 is set forth as NCBIRef. No. NM_005430.3, incorporated by reference herein as present in thedatabase on Dec. 1, 2103)

(SEQ ID NO: 37) gcggtgccgcccgccgtggccgcctcagcccaccagccgggaccgcgagccatgctgtccgccgcccgcccccagggttgttaaagccagactgcgaactctcgccactgccgccaccgccgcgtcccgtcccaccgtcgcgggcaacaaccaaagtcgccgcaactgcagcacagagcgggcaaagccaggcaggccatggggctctgggcgctgttgcctggctgggtttctgctacgctgctgctggcgctggccgctctgcccgcagccctggctgccaacagcagtggccgatggtggggtattgtgaacgtagcctcctccacgaacctgcttacagactccaagagtctgcaactggtactcgagcccagtctgcagctgttgagccgcaaacagcggcgtctgatacgccaaggaatcgccgctggaactgtcccactgctccagggccccacctcttcggcaagatcgtcaaccgaggctgtcgagaaacggcgtttatcttcgctatcacctccgccggggtcacccattcggtggcgcgctcctgctcagaaggttccatcgaatcctgcacgtgtgactaccggcggcgcggccccgggggccccgactggcactgggggggctgcagcgacaacattgacttcggccgcctcttcggccgggagttcgtggactccggggagaaggggcgggacctgcgcttcctcatgaaccttcacaacaacgaggcaggccgtacgaccgtattctccgagatgcgccaggagtgcaagtgccacgggatgtccggctcatgcacggtgcgcacgtgctggatgcggctgcccacgctgcgcgccgtgggcgatgtgctgcgcgaccgcttcgacggcgcctcgcgcgtcctgtacggcaaccgcggcagcaaccgcgcttcgcgggcggagctgctgcgcctggagccggaagacccggcccacaaaccgccctccccccacgacctcgtctacttcgagaaatcgcccaacttctgcacgtacagcggacgcctgggcacagcaggcacggcagggcgcgcctgtaacagctcgtcgcccgcgctggacggctgcgagctgctctgctgcggcaggggccaccgcacgcgcacgcagcgcgtcaccgagcgctgcaactgcaccttccactggtgctgccacgtcagctgccgcaactgcacgcacacgcgcgtactgcacgagtgtctgtgaggcgctgcgcggactcgcccccaggaacgctctcctcgagccctcccccaaacagactcgctagcactcaagacccggttattcgcccacccgagtacctccagtcacactccccgcggttcatacgcatcccatctctcccacttcctcctacctggggactcctcaaaccacttgcctggggcggcatgaaccctcttgccatcctgatggacctgccccggacctacctccctccctctccgcgggagaccccttgttgcactgccccctgcttggccaggaggtgagagaaggatgggtcccctccgccatggggtcggctcctgatggtgtcattctgcctgctccatcgcgccagcgacctctctgcctctcttcttcccctttgtcctgcgttttctccgggtcctcctaagtcccttcctattctcctgccatgggtgcagaccctgaacccacacctgggcatcagggcctttctcctccccacctgtagctgaagcaggaggttacagggcaaaagggcagctgtgatgatgtggaaatgaggttgggggaaccagcagaaatgcccccattctcccagtctctgtcgtggagccattgaacagctgtgagccatgcctccctgggccacctcctaccccttcctgtcctgcctcctcatcagtgtgtaaataatttgcactgaaacgtggatacagagccacgagtttggatgttgtaaataaaactatttattgtgctgggtcccagcctggtttgcaaagaccacctccaacccaacccaatccctctccactcttctctcctttctccctgcagccttttctggtccctcttctctcctcagtttctcaaagatgcgtttgcctcctggaatcagtatttccttccactgtagctattagcggctcctcgcccccaccagtgtagcatcttcctctgcagaataa aatctctattttta

In some embodiments the antibody or antigen binding fragment (such as anantibody or antigen binding fragment including the CDRs of one of theclone 13, clone 22, or clone 26 matrilin-3 specific antibodies) isconjugated to a parathyroid hormone (PTH) protein or fragment thereofthat binds the type 1 PTH receptor. PTH binding to the type 1 PTHreceptor is known to suppress chondrocyte maturation and reducecartilage degradation in osteoporosis models (see, e.g., Sampson et al.,Sci. Transl. Med., 3:101ra93, 2011). Exemplary PTH polypeptide andnucleic acid sequences are known to the person of ordinary skill in theart. An exemplary polypeptide sequence of a PTH protein is set forth asare available as NCBI Ref. No. NP_000306.1, which is incorporated byreference herein as present in the database on Jan. 3, 2015, andprovides as:

(SEQ ID NO: 50) KSVKKRSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ

Mature forms of PTH can also be used in a disclosed conjugate, such as

(SEQ ID NO: 51) KSVKKRSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ

In additional embodiments, the “teriparatide” sequence of PTH (residues1-34) can be used in a disclosed conjugate, which is set forth as:SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF (SEQ ID NO: 52).

An exemplary nucleic acid sequence encoding a PTH protein is set forthas NCBI Ref. No. NM_000315.2, incorporated by reference herein aspresent in the database on Jan. 3, 2015), and provided as

(SEQ ID NO: 53) Aaaagtcaccatttaaggggtctgcagtccaattcatcagttgtctttagtttactcagcatcagctactaacatacctgaacgaagatcttgttctaagacattgtatgtgaagatgatacctgcaaaagacatggctaaagttatgattgtcatgttggcaatttgttttcttacaaaatcggatgggaaatctgttaagaagagatctgtgagtgaaatacagcttatgcataacctgggaaaacatctgaactcgatggagagagtagaatggctgcgtaagaagctgcaggatgtgcacaattttgttgcccttggagctcctctagctcccagagatgctggttcccagaggccccgaaaaaaggaagacaatgtcttggttgagagccatgaaaaaagtcttggagaggcagacaaagctgatgtgaatgtattaactaaagctaaatcccagtgaaaatgaaaacagatattgtcagagttctgctctagacagtgtagggcaacaatacatgctgctaattcaaagctctattaagatttccaagtgccaatatttctgatataacaaactacatgtaatccatcactagccatgataactgcaattttaattgattattctgattccacttttattcatttgagttattttaattatcttttctattgtttattctttttaaagtatgttattgcataatttataaaagaataaaattgcacttttaaacctctcttctaccttaaaatgtaaaacaaaaatgtaatgatcataagtctaaataaatgaagtatttctcactcaaaaaaaaaaaaaaa

In some embodiments, conjugates of an antibody or antigen bindingfragment and one or more anti-inflammatory agents are provided.Non-limiting examples of such agents include dexamethasone, prednisone,and prednisolone. These conjugates inhibit chondrogenesis, and thereforeare not useful as chondrogenic agents, but can be used, for example, fortreatment of inflammation associated with arthritis. In additionalembodiments, conjugates of an antibody or antigen binding fragment andone or more agents that inhibit inflammation and/or local immuneresponse (such as an anti-arthritis agent), are provided. Non-limitingexamples of such agents include dexamethasone, prednisone, prednisolone,etanercept, adalimumab, infliximab, rituximab, anakinra. These agentscan be used, for example, for the treatment of auto-immune disordersaffecting cartilage, such as rheumatoid arthritis.

Conjugates including the antibody or antigen binding fragment linked toone or more anti-arthritis agents or chondrogenic agents (such as agrowth-regulating endocrine signaling molecule (e.g., a growth hormoneor a IGF-1), a growth-regulating paracrine signaling molecule (such asan IHH, a BMP, a CNP, a Wnt, or a FGF), or a steroid (e.g., an estrogen,an androgen, estradiol), can be produced according to known methods. Inone non-limiting example, a nucleic acid molecule encoding achondrogenic agent (such as a growth-regulating endocrine signalingmolecule (e.g., growth hormone or IGF-1), or a growth-regulatingparacrine signaling molecules (such as an IHH, a BMP, a CNP, a Wnt, or aFGF) is operably linked to a nucleic acid molecule encoding the antibodyor antigen binding fragment, for example and antibody or antigen bindingfragment that specifically binds to matrilin-3, such as an antibody orantigen binding fragment including the CDRs of the clone 13, clone 22,or clone 26 antibodies. In the case of an scFv, the effector moleculecan be linked to the N- and/or C-terminus of the scFv, for example. Inthe case of an IgG, the effector molecule can be linked to the N- and/orC-terminus of the heavy or light chain of the IgG. The nucleic acidmolecule can encode one or more chondrogenic agents, which can be linkedin series to the antibody or antigen binding fragment. Expression of thenucleic acid molecules under suitable conditions can be used to producethe conjugate.

In further embodiments an antibody or matrilin-3 binding fragmentthereof (such as an antibody or antigen binding fragment that includesthe CDRs of the clone 13, clone 22, or clone 26 matrilin-3 specificantibodies as disclosed herein) is conjugated to a steroid, such asdexamethasone or estradiol. Conjugating estradiol to peptides that bindto bone matrix has been shown to successfully deliver the steroidhormone to bone tissue in mice, with a marked increase in the retentiontime, producing biological effects on bone tissue while minimizingeffects on non-skeletal tissues (Yokogawa, et al. Endocrinology 142,1228-1233, 2001).

The antibody or antigen binding fragment can also be conjugated with adetectable marker; for example, a detectable marker capable of detectionby ELISA, spectrophotometry, flow cytometry, microscopy or diagnosticimaging techniques (such as computed tomography (CT), computed axialtomography (CAT) scans, magnetic resonance imaging (MRI), nuclearmagnetic resonance imaging NMRI), magnetic resonance tomography (MTR),ultrasound, fiberoptic examination, and laparoscopic examination).Specific, non-limiting examples of detectable markers includefluorophores, chemiluminescent agents, enzymatic linkages, radioactiveisotopes and heavy metals or compounds (for example super paramagneticiron oxide nanocrystals for detection by MRI). For example, usefuldetectable markers include fluorescent compounds, including fluorescein,fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanidephosphors and the like. Bioluminescent markers are also of use, such asluciferase, Green fluorescent protein (GFP), Yellow fluorescent protein(YFP). An antibody or antigen binding fragment can also be conjugatedwith enzymes that are useful for detection, such as horseradishperoxidase, β-galactosidase, luciferase, alkaline phosphatase, glucoseoxidase and the like. When an antibody or antigen binding fragment isconjugated with a detectable enzyme, it can be detected by addingadditional reagents that the enzyme uses to produce a reaction productthat can be discerned. For example, when the agent horseradishperoxidase is present the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which is visuallydetectable. An antibody or antigen binding fragment may also beconjugated with biotin, and detected through indirect measurement ofavidin or streptavidin binding. It should be noted that the avidinitself can be conjugated with an enzyme or a fluorescent label.

The antibody or antigen binding fragment can be conjugated with aparamagnetic agent, such as gadolinium. Paramagnetic agents such assuperparamagnetic iron oxide are also of use as labels. Antibodies canalso be conjugated with lanthanides (such as europium and dysprosium),and manganese. An antibody or antigen binding fragment may also belabeled with a predetermined polypeptide epitopes recognized by asecondary reporter (such as leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags).

The antibody or antigen binding fragment can also be conjugated with aradiolabeled amino acid. The radiolabel may be used for both diagnosticand therapeutic purposes. For instance, the radiolabel may be used todetect matrilin-3 and matrilin-3 expressing cells by x-ray, emissionspectra, or other diagnostic techniques. Examples of labels forpolypeptides include, but are not limited to, the followingradioisotopes or radionucleotides: ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In,¹²⁵I, ¹³¹I.

Means of detecting such detectable markers are well known to those ofskill in the art. Thus, for example, radiolabels may be detected usingphotographic film or scintillation counters, fluorescent markers may bedetected using a photodetector to detect emitted illumination. Enzymaticlabels are typically detected by providing the enzyme with a substrateand detecting the reaction product produced by the action of the enzymeon the substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

The average number of effector molecule or detectable marker moietiesper antibody or antigen binding fragment in a conjugate can range, forexample, from 1 to 20 moieties per antibody or antigen binding fragment.In certain embodiments, the average number of effector molecule ordetectable marker moieties per antibody or antigen binding fragment in aconjugate range from about 1 to about 2, from about 1 to about 3, about1 to about 8; from about 2 to about 6; from about 3 to about 5; or fromabout 3 to about 4. The loading (for example, effector molecule/antibodyratio) of an conjugate may be controlled in different ways, for example,by: (i) limiting the molar excess of effector molecule-linkerintermediate or linker reagent relative to antibody, (ii) limiting theconjugation reaction time or temperature, (iii) partial or limitingreductive conditions for cysteine thiol modification, (iv) engineeringby recombinant techniques the amino acid sequence of the antibody suchthat the number and position of cysteine residues is modified forcontrol of the number or position of linker-effector moleculeattachments.

C. Polynucleotides and Expression

Nucleic acid molecules (also referred to as polynucleotides) encodingthe polypeptides provided herein (including, but not limited toantibodies, antigen binding fragments and conjugates) can readily beproduced by one of skill in the art. For example, these nucleic acidscan be produced using the amino acid and nucleic acid sequences providedherein (such as the CDR sequences, heavy chain and light chain sequencesand sequences of chondrogenic agents).

One of skill in the art can readily use the genetic code to construct avariety of functionally equivalent nucleic acids, such as nucleic acidswhich differ in sequence but which encode the same antibody sequence, orencode a conjugate or fusion protein including the V_(L) and/or V_(H)nucleic acid sequence.

The nucleic acid molecules can be prepared by any suitable methodincluding, for example, cloning of appropriate sequences or by directchemical synthesis by methods such as the phosphotriester method ofNarang et al., Meth. Enzymol. 68:90-99, 1979; the phosphodiester methodof Brown et al., Meth. Enzymol. 68:109-151, 1979; thediethylphosphoramidite method of Beaucage et al., Tetra. Lett.22:1859-1862, 1981; the solid phase phosphoramidite triester methoddescribed by Beaucage & Caruthers, Tetra. Letts. 22(20):1859-1862, 1981,for example, using an automated synthesizer as described in, forexample, Needham-VanDevanter et al., Nucl. Acids Res. 12:6159-6168,1984; and, the solid support method of U.S. Pat. No. 4,458,066. Chemicalsynthesis produces a single stranded oligonucleotide. This can beconverted into double stranded DNA by hybridization with a complementarysequence or by polymerization with a DNA polymerase using the singlestrand as a template.

Exemplary nucleic acids can be prepared by cloning techniques. Examplesof appropriate cloning and sequencing techniques, and instructionssufficient to direct persons of skill through many cloning exercises areknown (see, e.g, Sambrook et al. (Molecular Cloning: A LaboratoryManual, 4^(th) ed, Cold Spring Harbor, New York, 2012) and Ausubel etal. (In Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, through supplement 104, 2013). Product information frommanufacturers of biological reagents and experimental equipment alsoprovide useful information. Such manufacturers include the SIGMAChemical Company (Saint Louis, MO), R&D Systems (Minneapolis, MN),Pharmacia Amersham (Piscataway, NJ), CLONTECH Laboratories, Inc. (PaloAlto, CA), Chem Genes Corp., Aldrich Chemical Company (Milwaukee, WI),Glen Research, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersburg,MD), Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs,Switzerland), Invitrogen (Carlsbad, CA), and Applied Biosystems (FosterCity, CA), as well as many other commercial sources known to one ofskill.

Nucleic acids can also be prepared by amplification methods.Amplification methods include polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR). Awide variety of cloning methods, host cells, and in vitro amplificationmethodologies are well known to persons of skill.

The nucleic acid molecules can be expressed in a recombinantlyengineered cell such as bacteria, plant, yeast, insect and mammaliancells. The antibodies antigen binding fragments, and conjugates can beexpressed as individual V_(H) and/or V_(L) chain (linked to an effectormolecule or detectable marker as needed), or can be expressed as afusion protein. Methods of expressing and purifying antibodies andantigen binding fragments are known and further described herein (see,e.g., Al-Rubeai (ed), Antibody Expression and Production, SpringerPress, 2011). An immunoadhesin can also be expressed. Thus, in someexamples, nucleic acids encoding a V_(H) and V_(L), and immunoadhesinare provided. The nucleic acid sequences can optionally encode a leadersequence.

To create a scFv the V_(H)- and V_(L)-encoding DNA fragments can beoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄-Ser)₃, such that the V_(H) andV_(L) sequences can be expressed as a contiguous single-chain protein,with the V_(L) and V_(H) domains joined by the flexible linker (see,e.g., Bird et al., Science 242:423-426, 1988; Huston et al., Proc. Natl.Acad. Sci. USA 85:5879-5883, 1988; McCafferty et al., Nature348:552-554, 1990; Kontermann and Dubel (Ed), Antibody Engineering,Vols. 1-2, 2^(nd) Ed., Springer Press, 2010; Harlow and Lane,Antibodies: A Laboratory Manual, 2^(nd), Cold Spring Harbor Laboratory,New York, 2013). Optionally, a cleavage site can be included in alinker, such as a furin cleavage site.

The nucleic acid encoding the V_(H) and/or the V_(L) optionally canencode an Fc domain (immunoadhesin). The Fc domain can be an IgA, IgM orIgG Fc domain. The Fc domain can be an optimized Fc domain, as describedin U.S. Published Patent Application No. 20100/093979, incorporatedherein by reference. In one example, the immunoadhesin is an IgG1 Fc. Inone example, the immunoadhesin is an IgG3 Fc.

The single chain antibody may be monovalent, if only a single V_(H) andV_(L) are used, bivalent, if two V_(H) and V_(L) are used, orpolyvalent, if more than two V_(H) and V_(L) are used. Bispecific orpolyvalent antibodies may be generated that bind specifically tomatrilin-3 and to another molecule, such as a chondrogenesis biomarker.The encoded V_(H) and V_(L) optionally can include a furin cleavage sitebetween the V_(H) and V_(L) domains.

Those of skill in the art are knowledgeable in the numerous expressionsystems available for expression of proteins including E. coli, otherbacterial hosts, yeast, and various higher eukaryotic cells such as theCOS, CHO, HeLa and myeloma cell lines.

One or more DNA sequences encoding the antibody or fragment thereof canbe expressed in vitro by DNA transfer into a suitable host cell. Thecell may be prokaryotic or eukaryotic. The term also includes anyprogeny of the subject host cell. It is understood that all progeny maynot be identical to the parental cell since there may be mutations thatoccur during replication. Methods of stable transfer, meaning that theforeign DNA is continuously maintained in the host, are known in theart. Hybridomas expressing the antibodies of interest are alsoencompassed by this disclosure.

The expression of nucleic acids encoding the proteins described hereincan be achieved by operably linking the DNA or cDNA to a promoter (whichis either constitutive or inducible), followed by incorporation into anexpression cassette. The promoter can be any promoter of interest,including a cytomegalovirus promoter and a human T cell lymphotrophicvirus promoter (HTLV)-1. Optionally, an enhancer, such as acytomegalovirus enhancer, is included in the construct. The cassettescan be suitable for replication and integration in either prokaryotes oreukaryotes. Typical expression cassettes contain specific sequencesuseful for regulation of the expression of the DNA encoding the protein.For example, the expression cassettes can include appropriate promoters,enhancers, transcription and translation terminators, initiationsequences, a start codon (i.e., ATG) in front of a protein-encodinggene, splicing signal for introns, sequences for the maintenance of thecorrect reading frame of that gene to permit proper translation of mRNA,and stop codons. The vector can encode a selectable marker, such as amarker encoding drug resistance (for example, ampicillin or tetracyclineresistance).

To obtain high level expression of a cloned gene, it is desirable toconstruct expression cassettes which contain, at the minimum, a strongpromoter to direct transcription, a ribosome binding site fortranslational initiation (internal ribosomal binding sequences), and atranscription/translation terminator. For E. coli, this can include apromoter such as the T7, trp, lac, or lambda promoters, a ribosomebinding site, and preferably a transcription termination signal. Foreukaryotic cells, the control sequences can include a promoter and/or anenhancer derived from, for example, an immunoglobulin gene, HTLV, SV40or cytomegalovirus, and a polyadenylation sequence, and can furtherinclude splice donor and/or acceptor sequences (for example, CMV and/orHTLV splice acceptor and donor sequences). The cassettes can betransferred into the chosen host cell by well-known methods such astransformation or electroporation for E. coli and calcium phosphatetreatment, electroporation or lipofection for mammalian cells. Cellstransformed by the cassettes can be selected by resistance toantibiotics conferred by genes contained in the cassettes, such as theamp, gpt, neo and hyg genes.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with polynucleotide sequences encoding the antibody,labeled antibody, or matrilin-3 binding fragment thereof, and a secondforeign DNA molecule encoding a selectable phenotype, such as the herpessimplex thymidine kinase gene. Another method is to use a eukaryoticviral vector, such as simian virus (SV40) or bovine papilloma virus, totransiently infect or transform eukaryotic cells and express the protein(see for example, Viral Expression Vectors, Springer press, Muzyczkaed., 2011). One of skill in the art can readily use an expressionsystems such as plasmids and vectors of use in producing proteins incells including higher eukaryotic cells such as the COS, CHO, HeLa andmyeloma cell lines.

Modifications can be made to a nucleic acid encoding a polypeptidedescribed herein without diminishing its biological activity. Somemodifications can be made to facilitate the cloning, expression, orincorporation of the targeting molecule into a fusion protein. Suchmodifications are well known to those of skill in the art and include,for example, termination codons, a methionine added at the aminoterminus to provide an initiation site, additional amino acids placed oneither terminus to create conveniently located restriction sites, oradditional amino acids (such as poly His) to aid in purification steps.In addition to recombinant methods, the antibodies, antigen bindingfragments, and conjugates can also be constructed in whole or in partusing standard peptide synthesis well known in the art.

Once expressed, the antibodies, antigen binding fragments, andconjugates can be purified according to standard procedures in the art,including ammonium sulfate precipitation, affinity columns, columnchromatography, and the like (see, generally, Simpson ed., Basic methodsin Protein Purification and Analysis: A laboratory Manual, Cold HarborPress, 2008). The antibodies, antigen binding fragment, and conjugatesneed not be 100% pure. Once purified, partially or to homogeneity asdesired, if to be used therapeutically, the polypeptides should besubstantially free of endotoxin.

Methods for expression of the antibodies, antigen binding fragments, andconjugates, and/or refolding to an appropriate active form, frommammalian cells, and bacteria such as E. coli have been described andare well-known and are applicable to the antibodies disclosed herein.See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, 2^(nd),Cold Spring Harbor Laboratory, New York, 2013, Simpson ed., Basicmethods in Protein Purification and Analysis: A laboratory Manual, ColdHarbor Press, 2008, and Ward et al., Nature 341:544, 1989.

In addition to recombinant methods, the antibodies, antigen bindingfragments, and/or conjugates can also be constructed in whole or in partusing standard peptide synthesis. Solid phase synthesis of thepolypeptides can be accomplished by attaching the C-terminal amino acidof the sequence to an insoluble support followed by sequential additionof the remaining amino acids in the sequence. Techniques for solid phasesynthesis are described by Barany & Merrifield, The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA. pp. 3-284; Merrifield et al., J. Am. Chem. Soc. 85:2149-2156, 1963,and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem.Co., Rockford, Ill., 1984. Proteins of greater length may be synthesizedby condensation of the amino and carboxyl termini of shorter fragments.Methods of forming peptide bonds by activation of a carboxyl terminalend (such as by the use of the coupling reagent N,N′-dicyclohexylcarbodiimide) are well known in the art.

E. Therapeutic Methods, Methods of Detection, and Compositions

A therapeutically effective amount of an antibody or antigen bindingfragment that specifically binds matrilin-3, or conjugate thereof (suchas a conjugate including a chondrogenic agent or anti-arthritis agent),or a nucleic acid molecule or vector encoding such a molecule, or acomposition including such molecules, can be administered to a subjectto increase height and/or to treat or inhibit a cartilage disorderand/or treat or inhibit arthritis (such as rheumatoid arthritis) in asubject, for example to treat or inhibit a growth plate cartilagedisorder (such as a skeletal dysplasia, or short stature) or anarticular cartilage disorder (such as osteoarthritis). In theseapplications, a therapeutically effective amount of the antibody,antigen binding fragment, or conjugate (e.g., that specifically bindsmatrilin-3), or a nucleic acid molecule or vector encoding such amolecule, or a composition including such molecules is administered to asubject in an amount and under conditions sufficient to form an immunecomplex with matrilin-3, thereby treating or inhibiting the cartilagedisorder in the subject, for example, by increasing chondrogenesis incartilage of the subject (such as growth plate cartilage or articularcartilage). A subject can be selected for treatment that has, issuspected of having, or is at risk of developing, a cartilage disorder,such as a growth plate cartilage disorder (e.g., a skeletal dysplasia,or short stature) or an articulate cartilage disorder (e.g.,osteoarthritis). Subjects that can benefit from the disclosed methodsinclude human and veterinary subjects.

The therapeutically effective amount will depend upon the severity ofthe disease and the general state of the patient's health. Atherapeutically effective amount is that which provides eithersubjective relief of a symptom(s) or an objectively identifiableimprovement as noted by the clinician or other qualified observer. Inone embodiment, a therapeutically effective amount is the amountnecessary to increase chondrogenesis in the cartilage (such as growthplate cartilage or articular cartilage) of a subject, or the amount thateffectively treats or inhibits, or effectively reduces a sign or asymptom of, a cartilage disorder in a subject (such as a skeletaldysplasia, short stature, or osteoarthritis). The therapeuticallyeffective amount of the agents administered can vary depending upon thedesired effects and the subject to be treated.

The cartilage disorder or arthritis does not need to be completelyeliminated for the method to be effective. For example, a disclosedmethod of treating or inhibiting a cartilage disorder or arthritis candecrease the cartilage disorder or arthritis by a desired amount, forexample by at least 10%, at least 20%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, oreven at least 100% (elimination of all signs and symptoms of thecartilage disorder or arthritis), as compared to the cartilage disorderor arthritis in the absence of the treatment.

In some embodiments, a disclosed method of treating or inhibiting acartilage disorder can increase the longitudinal growth of a subjectwith a cartilage disorder as compared the longitudinal growth in theabsence of the treatment, such as an increase of at least at least 10%,at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 100%, at least 150%, at least 200%, at least 300%,at least 400, or at least 500% in the longitudinal growth of the subjectas compared to the longitudinal growth in the absence of the treatment.

In some embodiments, a method of increasing chondrogenesis in cartilagetissue is provided. The methods include contacting the cartilage tissuewith a therapeutically effective amount of an antibody or antigenbinding fragment that specifically binds matrilin-3, or conjugatethereof (such as a conjugate including a chondrogenic agent), or anucleic acid molecule or vector encoding such a molecule, or acomposition including such molecules. In one non-limiting example, thecartilage tissue is contacted with a conjugate including a chondrogenicagent linked to an antibody or antigen binding fragment thatspecifically binds matrilin-3. The cartilage tissue can also becontacted with one or more additional agents, such as described hereinfor combination therapies. The cartilage tissue can be in vivo or invitro. In some embodiments, chondrogenesis in the cartilage tissue isincreased by at least 10%, at least 20%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 100%, at least 200%, atleast 300%, at least 400% or even at least 500% compared to controlconditions.

Also provided is a method of detecting matrilin-3 expression in vitro orin vivo. In one example, expression of matrilin-3 is detected in abiological sample, and can be used to detect cartilage tissue in asubject or in a sample. The sample can be any sample, including, but notlimited to, tissue from biopsies, autopsies and pathology specimens.Biological samples also include sections of tissues, for example, frozensections taken for histological purposes. Biological samples furtherinclude body fluids, such as blood, serum, plasma, sputum, spinal fluidor urine. The method of detection can include contacting a cell orsample, or administering to a subject, an antibody or antigen bindingfragment that specifically binds to matrilin-3, or conjugate there of(e.g. a conjugate including a detectable marker) under conditionssufficient to form an immune complex, and detecting the immune complex(e.g., by detecting a detectable marker conjugated to the antibody orantigen binding fragment.

Dosages and Frequency of Dosing

A therapeutically effective amount of an antibody or antigen bindingfragment that specifically binds matrilin-3, or conjugate thereof (suchas a conjugate including a chondrogenic agent or anti-arthritis agent),or a nucleic acid molecule or vector encoding such a molecule, or acomposition including such molecules, can be readily ascertained by oneskilled in the art, using publicly available materials and procedures.For example, the amount of an agent used for therapy should induce anincrease in chondrogenesis and/or longitudinal growth of the patientcompared to control conditions. In some embodiments, the therapy inducesan increase in longitudinal growth compared to established growthpatterns of children ages 0-17 years with a skeletal dysplasia, or shortstature, or other cartilage disorder. Established growth patterns ofsuch subjects are known, and can be obtained, for example, fromestablished height for age, head circumference, and segmental growth(Horton et al., Standard growth curves for achondroplasia, J. Pediatr.,93: 435-8 (1978, incorporated by reference herein)).

The dosing frequency for a particular individual may vary depending uponvarious factors, including the disorder being treated and the conditionand response of the individual to the therapy. In certain embodiments, apharmaceutical composition containing the antibody or antigen bindingfragment that specifically binds matrilin-3, or conjugate thereof (suchas a conjugate including a chondrogenic agent), or a nucleic acidmolecule or vector encoding such a molecule, is administered to asubject about one time per day, one time per two days, one time perthree days, or one time per week. In one embodiment, for treatment ofcartilage disorders (e.g., skeletal dysplasias, including achondroplasiaand short stature), a daily or weekly dose of such therapeutic agents isadministered to patients until and/or through adulthood.

Data obtained from cell culture assays and animal studies can be used toformulate a range of dosage for use in humans. The dosage normally lieswithin a range of circulating concentrations that include the ED₅₀, withlittle or minimal toxicity. The dosage can vary within this rangedepending upon the dosage form employed and the route of administrationutilized. The therapeutically effective dose can be determined from cellculture assays and animal studies. In certain embodiments, the antibodyor antigen binding fragment that specifically binds matrilin-3, orconjugate thereof (such as a conjugate including a chondrogenic agent),or a nucleic acid molecule or vector encoding such a molecule, or acomposition including such molecules, is administered at a dose in therange of from about 5 or 10 nmol/kg to about 300 nmol/kg, or from about20 nmol/kg to about 200 nmol/kg, or at a dose of about 5, 10, 15, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110,120, 125, 130, 140, 150, 160, 170, 175, 180, 190, 200, 210, 220, 230,240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 750, 1000, 1250,1500, 1750 or 2000 nmol/kg, or at a dose of about 5, 10, 20, 30, 40, 50,60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950 or 1000 ug/kg, or about 1, 1.25, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5,5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mg/kg, or other dose deemedappropriate by the treating physician. The doses described herein can beadministered according to the dosing frequency/frequency ofadministration described herein, including without limitation daily, 2or 3 times per week, weekly, every 2 weeks, every 3 weeks, monthly, etc.

In some embodiments, a disclosed therapeutic agent is administered so asto allow for periods of growth (e.g., chondrogenesis), followed by arecovery period (e.g., osteogenesis). For example, the therapeutic agentmay be administered intravenously, subcutaneously or by another modedaily or multiple times per week for a period of time, followed by aperiod of no treatment, then the cycle is repeated. In some embodiments,the initial period of treatment (e.g., administration of the therapeuticagent daily or multiple times per week) is for 3 days, 1 week, 2 weeks,3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks,11 weeks or 12 weeks. In a related embodiment, the period of notreatment lasts for 3 days, 1 week, 2 weeks, 3 weeks or 4 weeks. Incertain embodiments, the dosing regimen of the therapeutic agent isdaily for 3 days followed by 3 days off; or daily or multiple times perweek for 1 week followed by 3 days or 1 week off; or daily or multipletimes per week for 2 weeks followed by 1 or 2 weeks off; or daily ormultiple times per week for 3 weeks followed by 1, 2 or 3 weeks off; ordaily or multiple times per week for 4, 5, 6, 7, 8, 9, 10, 11 or 12weeks followed by 1, 2, 3 or 4 weeks off.

Modes of Administration

An antibody or antigen binding fragment that specifically bindsmatrilin-3, or conjugate thereof (such as a conjugate including achondrogenic agent or anti-arthritis agent), or a nucleic acid moleculeor vector encoding such a molecule, or a composition including suchmolecules, as well as additional agents, can be administered to subjectsin various ways, including local and systemic administration, such as,e.g., by injection subcutaneously, intravenously, intra-arterially,intraperitoneally, intramuscularly, intradermally, or intrathecally. Inan embodiment, a therapeutic agent are administered by a singlesubcutaneous, intravenous, intra-arterial, intraperitoneal,intramuscular, intradermal or intrathecal injection once a day.

The therapeutic agent can also be administered by direct injection at ornear the site of disease. Further, the therapeutic agent can beadministered by implantation of a depot at the target site of action(e.g., an abnormal or dysplasic cartilage). The therapeutic agent mayalso be administered orally in the form of microspheres, microcapsules,liposomes (uncharged or charged (e.g., cationic)), polymericmicroparticles (e.g., polyamides, polylactide, polyglycolide,poly(lactide-glycolide)), microemulsions, and the like.

A further method of administration is by osmotic pump (e.g., an Alzetpump) or mini-pump (e.g., an Alzet mini-osmotic pump), which allows forcontrolled, continuous and/or slow-release delivery of the therapeuticagent or pharmaceutical composition over a pre-determined period. Theosmotic pump or mini-pump can be implanted subcutaneously, or near thetarget site (e.g., the long bones of limbs, the growth plate, etc.). Itwill be apparent to one skilled in the art that the therapeutic agent orcompositions thereof can also be administered by other modes.Determination of the most effective mode of administration of thetherapeutic agent or compositions thereof is within the skill of theskilled artisan. The therapeutic agent can be administered aspharmaceutical formulations suitable for, e.g., oral (including buccaland sub-lingual), rectal, nasal, topical, pulmonary, vaginal orparenteral (including intramuscular, intraarterial, intrathecal,subcutaneous and intravenous) administration, or in a form suitable foradministration by inhalation or insufflation. Depending on the intendedmode of administration, the pharmaceutical formulations can be in theform of solid, semi-solid or liquid dosage forms, such as tablets,suppositories, pills, capsules, powders, liquids, suspensions,emulsions, creams, ointments, lotions, and the like. The formulationscan be provided in unit dosage form suitable for single administrationof a precise dosage. The formulations comprise an effective amount of atherapeutic agent, and one or more pharmaceutically acceptableexcipients, carriers and/or diluents, and optionally one or more otherbiologically active agents.

Combination Therapy

In one embodiment, the antibody or antigen binding fragment thatspecifically binds matrilin-3, or conjugate thereof (such as a conjugateincluding a chondrogenic agent), or a nucleic acid molecule or vectorencoding such a molecule, or a composition including such molecules canbe used in combination with one or more other active agents useful fortreating, ameliorating or preventing a cartilage disorder such as, e.g.,short stature. The other active agent(s) can enhance the effects of thetherapeutic agent and/or exert other pharmacological effects in additionto those of the therapeutic agent. Non-limiting examples of activeagents that can be used in combination with the therapeutic agentdescribed herein are natriuretic peptides (e.g., BNP) and inhibitors(e.g., antagonists) of peptidases and proteases (e.g., NEP and furin),NPR-C and tyrosine kinases (e.g., FGFR-3).

Co-use of a tyrosine kinase inhibitor can accentuate the therapeuticeffects by inhibiting the tyrosine kinase receptor FGFR-3, a negativeregulator of chondrogenesis. Non-limiting examples of tyrosine kinaseinhibitors include those disclosed in U.S. Pat. Nos. 6,329,375 and6,344,459, which are incorporated by reference herein.

To achieve the appropriate therapeutic outcome in the combinationtherapies, one would generally administer to the subject the antibody orantigen binding fragment that specifically binds matrilin-3, orconjugate thereof (such as a conjugate including a chondrogenic agent),or a nucleic acid molecule or vector encoding such a molecule, or acomposition including such molecules and other therapeutic(s) in acombined amount effective to produce the desired therapeutic outcome(e.g., chondrogenesis and/or increased linear growth). This process mayinvolve administering the antibody or antigen binding fragment thatspecifically binds matrilin-3, or conjugate thereof (such as a conjugateincluding a chondrogenic agent), or a nucleic acid molecule or vectorencoding such a molecule, or a composition including such molecules, andother therapeutic agent(s) at the same time. Simultaneous administrationcan be achieved by administering a single composition or pharmacologicalformulation that includes multiple agent(s). Alternatively, the othertherapeutic agent(s) can be taken separately at about the same time as apharmacological formulation (e.g., tablet, injection or drink) of theantibody or antigen binding fragment that specifically binds matrilin-3,or conjugate thereof (such as a conjugate including a chondrogenicagent), or a nucleic acid molecule or vector encoding such a molecule,or a composition including such molecules.

In other alternatives, administration of the antibody or antigen bindingfragment that specifically binds matrilin-3, or conjugate thereof (suchas a conjugate including a chondrogenic agent), or a nucleic acidmolecule or vector encoding such a molecule, or a composition includingsuch molecules can precede or follow administration of the othertherapeutic agent(s) by intervals ranging from minutes to hours. Inembodiments where the other therapeutic agent(s) and the antibody orantigen binding fragment that specifically binds matrilin-3, orconjugate thereof (such as a conjugate including a chondrogenic agent),or a nucleic acid molecule or vector encoding such a molecule, or acomposition including such molecules are administered separately, onewould generally ensure that the multiple agents are administered withinan appropriate time of one another so that each agent can exert,synergistically or additively, a beneficial effect on the patient. Forexample, one can administer the antibody or antigen binding fragmentthat specifically binds matrilin-3, or conjugate thereof (such as aconjugate including a chondrogenic agent), or a nucleic acid molecule orvector encoding such a molecule, or a composition including suchmolecules within about 0.5-6 hours (before or after) of the othertherapeutic agent(s). In one embodiment, the antibody or antigen bindingfragment that specifically binds matrilin-3, or conjugate thereof (suchas a conjugate including a chondrogenic agent), or a nucleic acidmolecule or vector encoding such a molecule, or a composition includingsuch molecules is administered within about 1 hour (before or after) ofthe other therapeutic agent(s).

The combination therapy may provide synergy and prove synergistic, thatis, the effect achieved when the active ingredients used together isgreater than the sum of the effects that results from using thecompounds separately. A synergistic effect may be attained when theactive ingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined, unit dosage formulation; (2) delivered byalternation or in parallel as separate formulations; or (3) by someother regimen. When delivered in alternation, a synergistic effect maybe attained when the compounds are administered or deliveredsequentially, for example by different injections in separate syringes.In general, during alternation, an effective dosage of each activeingredient is administered sequentially, i.e. serially, whereas incombination therapy, effective dosages of two or more active ingredientsare administered together.

Identifying and Monitoring Patient Populations

Protocols can be established to identify subjects suitable for therapyusing the antibody or antigen binding fragment that specifically bindsmatrilin-3, or conjugate thereof (such as a conjugate including achondrogenic agent or an anti-arthritis agent), or a nucleic acidmolecule or vector encoding such a molecule, or a composition includingsuch molecules and to determine whether a given patient is responsive tosuch therapy. For example, for treatment of cartilage disorders,indicators of growth can be measured, such as height, long bone growthmeasurements in utero or postnatal. The ameliorating effect on skeletaldeformity can be assessed by physical examination and clinical imagingmethods, such as radiographs, CT scans, MRI scans, and ultrasounds. Inaddition, measurements of bone growth biomarkers such as CNP, cGMP,Collagen II, osteocalcin, and Proliferating Cell Nuclear Antigen (PCNA)may serve as a useful marker for efficacy.

Cartilage-specific analytes (or cartilage-associated markers) can alsobe measured to assess therapeutic efficacy. For example, fragments ofcleaved collagen type II are a cartilage-specific marker for cartilageturnover. Type II collagen is the major organic constituent of cartilageand fragments of type II collagen (cleaved collagen) are released intocirculation, and subsequently secreted into the urine, followingcartilage turnover. Cartilage turnover precedes new bone formation.

A biomarker for bone formation which can be measured is N-terminalpropeptides of type I procollagen (PINP). The synthesis of type Icollagen is an important step in bone formation, as type I collagen isthe major organic component in bone matrix. During collagen synthesis,propeptides are released from the procollagen molecule and can bedetected in serum. Other potential biomarkers for cartilage formationand growth include aggrecan chondroitin sulfate (cartilage-specificmarker for cartilage turnover), and propeptides of type TT collagen(cartilage-specific marker for cartilage formation).Cartilage-associated biomarkers can be measured, e.g., in serum fromefficacy/pharmacodynamic in vivo studies and from the conditioned mediaof ex vivo studies, using commercially available kits.

In one embodiment, the level of at least one cartilage-associatedbiomarker is assayed or measured in a subject that has been administeredthe antibody or antigen binding fragment that specifically bindsmatrilin-3, or conjugate thereof (such as a conjugate including achondrogenic agent), or a nucleic acid molecule or vector encoding sucha molecule, or a composition including such molecules in order tomonitor the effects of the therapeutic agent on bone and cartilageformation and growth in vivo. For example, an increase in the level ofat least one cartilage-associated biomarker may indicate thatadministration of the therapeutic agent has a positive effect onchondrogenesis or bone growth and is a useful treatment for skeletaldysplasias and other cartilage disorders. Exemplary bone- orcartilage-associated biomarkers include, but are not limited to, CNP(e.g, endogenous levels of CNP), cGMP, propeptides of collagen type IIand fragments thereof, collagen type II and fragments thereof,osteocalcin, proliferating cell nuclear antigen (PCNA), propeptides oftype I procollagen (PINP) and fragments thereof, collagen type I andfragments thereof, aggrecan chondroitin sulfate, and alkalinephosphatase.

In an embodiment, biomarkers are measured by obtaining a biologicalsample from a subject who will be administered, is being administered orhas been administered one or more of an antibody or antigen bindingfragment that specifically binds matrilin-3, or conjugate thereof (suchas a conjugate including a chondrogenic agent), or a nucleic acidmolecule or vector encoding such a molecule, or a composition includingsuch molecules. Biomarkers can be measured using techniques known in theart, including, but not limited to, Western Blot, enzyme linkedimmunosorbant assay (ELISA), and enzymatic activity assay. Thebiological sample can be blood, serum, urine, or other biologicalfluids.

Additional aspects and details of the disclosure will be apparent fromthe following examples, which are intended to be illustrative ratherthan limiting.

Compositions

Compositions are provided that include one or more antibodies, antigenbinding fragments (such as an antibody or antigen binding fragment thatspecifically binds to matrilin-3), or conjugates thereof (such as aconjugate including a chondrogenic agent or anti-arthritis agent linkedto an antibody or antigen binding fragment that specifically bindsmatrilin-3), or nucleic acid molecules or vectors encoding suchmolecules in a carrier (such as a pharmaceutically acceptable carrier).The compositions can be prepared in unit dosage forms for administrationto a subject. The amount and timing of administration are at thediscretion of the treating clinician to achieve the desired outcome. Thecompositions can be formulated for systemic (such as intravenus) orlocal (such as intra-cartilage) administration. In one example, adisclosed antibody, antigen binding fragment, or conjugate, isformulated for parenteral administration, such as intravenousadministration. Compositions including a conjugate, antibody or antigenbinding fragment as disclosed herein are of use, for example, for thetreatment of cartilage disorders as well as for the detection ofcartilage. In some examples, the compositions are useful for thetreatment of a cartilage disorder such as a skeletal dysplasia, shortstature, or osteoarthritis.

In some embodiments, the compositions comprise an antibody, antigenbinding fragment, or conjugate thereof, in at least about 70%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98% or 99% purity. In certain embodiments, thecompositions contain less than about 10%, 5%, 4%, 3%, 2%, 1% or 0.5% ofmacromolecular contaminants, such as other mammalian (e.g., human)proteins.

The compositions for administration can include a solution of theconjugate, antibody or antigen binding fragment dissolved in apharmaceutically acceptable carrier, such as an aqueous carrier. Avariety of aqueous carriers can be used, for example, buffered salineand the like. These solutions are sterile and generally free ofundesirable matter. These compositions may be sterilized byconventional, well known sterilization techniques. The compositions maycontain pharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, for example, sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate and the like. The concentration of antibody or antigen bindingfragment or conjugate in these formulations can vary widely, and will beselected primarily based on fluid volumes, viscosities, body weight andthe like in accordance with the particular mode of administrationselected and the subject's needs. Actual methods of preparing suchdosage forms are known, or will be apparent, to those skilled in theart.

A typical composition for intravenous administration includes about 0.01to about 30 mg/kg of antibody or antigen binding fragment or conjugateper subject per day (or the corresponding dose of a conjugate includingthe antibody or antigen binding fragment). Actual methods for preparingadministrable compositions will be known or apparent to those skilled inthe art and are described in more detail in such publications asRemington's Pharmaceutical Science, 19th ed., Mack Publishing Company,Easton, PA (1995). In some embodiments, the composition can be a liquidformulation including one or more antibodies, antigen binding fragments(such as an antibody or antigen binding fragment that specifically bindsto matrilin-3), or conjugates thereof (such as a conjugate including achondrogenic agent linked to an antibody or antigen binding fragmentthat specifically binds matrilin-3), or nucleic acid molecules orvectors encoding such molecules in a concentration range from about 0.1mg/ml to about 20 mg/ml, or from about 0.5 mg/ml to about 20 mg/ml, orfrom about 1 mg/ml to about 20 mg/ml, or from about 0.1 mg/ml to about10 mg/ml, or from about 0.5 mg/ml to about 10 mg/ml, or from about 1mg/ml to about 10 mg/ml.

Therapeutic agents such as antibodies, antigen binding fragments, orconjugates may be provided in lyophilized form and rehydrated withsterile water before administration, although they are also provided insterile solutions of known concentration. The antibody or antigenbinding fragment or conjugate solution is then added to an infusion bagcontaining 0.9% sodium chloride, USP, and in some cases administered ata dosage of from 0.5 to 15 mg/kg of body weight. Considerable experienceis available in the art in the administration of antibody or antigenbinding fragment and conjugate drugs; for example, antibody drugs havebeen marketed in the U.S. since the approval of RITUXAN® in 1997. Theagents can be administered by slow infusion, rather than in anintravenous push or bolus. In one example, a higher loading dose isadministered, with subsequent, maintenance doses being administered at alower level. For example, an initial loading dose of 4 mg/kg antibody orantigen binding fragment (or the corresponding dose of a conjugateincluding the antibody or antigen binding fragment) may be infused overa period of some 90 minutes, followed by weekly maintenance doses for4-8 weeks of 2 mg/kg infused over a 30 minute period if the previousdose was well tolerated.

Controlled release parenteral formulations can be made as implants, oilyinjections, or as particulate systems. For a broad overview of proteindelivery systems see, Banga, A. J., Therapeutic Peptides and Proteins:Formulation, Processing, and Delivery Systems, Technomic PublishingCompany, Inc., Lancaster, PA, (1995). Particulate systems includemicrospheres, microparticles, microcapsules, nanocapsules, nanospheres,and nanoparticles. Microcapsules contain the therapeutic protein, suchas a cytotoxin or a drug, as a central core. In microspheres thetherapeutic is dispersed throughout the particle. Particles,microspheres, and microcapsules smaller than about 1 μm are generallyreferred to as nanoparticles, nanospheres, and nanocapsules,respectively. Capillaries have a diameter of approximately 5 μm so thatonly nanoparticles are administered intravenously. Microparticles aretypically around 100 μm in diameter and are administered subcutaneouslyor intramuscularly. See, for example, Kreuter, J., Colloidal DrugDelivery Systems, J. Kreuter, ed., Marcel Dekker, Inc., New York, NY,pp. 219-342 (1994); and Tice & Tabibi, Treatise on Controlled DrugDelivery, A. Kydonieus, ed., Marcel Dekker, Inc. New York, NY, pp.315-339, (1992).

Polymers can be used for ion-controlled release of the antibody orantigen binding fragment or conjugate compositions disclosed herein.Various degradable and nondegradable polymeric matrices for use incontrolled drug delivery are known in the art (Langer, Accounts Chem.Res. 26:537-542, 1993). For example, the block copolymer, polaxamer 407,exists as a viscous yet mobile liquid at low temperatures but forms asemisolid gel at body temperature. It has been shown to be an effectivevehicle for formulation and sustained delivery of recombinantinterleukin-2 and urease (Johnston et al., Pharm. Res. 9:425-434, 1992;and Pec et al., J. Parent. Sci. Tech. 44(2):58-65, 1990). Alternatively,hydroxyapatite has been used as a microcarrier for controlled release ofproteins (Ijntema et al., Int. J. Pharm. 112:215-224, 1994). In yetanother aspect, liposomes are used for controlled release as well asdrug targeting of the lipid-capsulated drug (Betageri et al., LiposomeDrug Delivery Systems, Technomic Publishing Co., Inc., Lancaster, PA(1993)). Numerous additional systems for controlled delivery oftherapeutic proteins are known (see U.S. Pat. Nos. 5,055,303; 5,188,837;4,235,871; 4,501,728; 4,837,028; 4,957,735; 5,019,369; 5,055,303;5,514,670; 5,413,797; 5,268,164; 5,004,697; 4,902,505; 5,506,206;5,271,961; 5,254,342 and 5,534,496).

F. Kits

Kits are also provided. For example, kits for treating a subject withone or more antibodies, antigen binding fragments (such as an antibodyor antigen binding fragment that specifically binds to matrilin-3), orconjugates thereof (such as a conjugate including a chondrogenic agentlinked to an antibody or antigen binding fragment that specificallybinds matrilin-3), nucleic acid molecules or vectors encoding suchmolecules, or compositions including such molecules. The kits willtypically include a disclosed antibody, antigen binding fragment,conjugate, nucleic acid molecule, or composition as disclosed herein.More than one of the disclosed antibodies, antigen binding fragments,conjugates, nucleic acid molecules, or compositions can be included inthe kit.

The kit can include a container and a label or package insert on orassociated with the container. Suitable containers include, for example,bottles, vials, syringes, etc. The containers may be formed from avariety of materials such as glass or plastic. The container typicallyholds a composition including one or more of the disclosed antibodies,antigen binding fragments, conjugates, nucleic acid molecules, orcompositions. In several embodiments the container may have a sterileaccess port (for example the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). A label or package insert indicates that the composition isused for treating the particular condition.

The label or package insert typically will further include instructionsfor use of the antibodies, antigen binding fragments, conjugates,nucleic acid molecules, or compositions included in the kit. The packageinsert typically includes instructions customarily included incommercial packages of therapeutic products that contain informationabout the indications, usage, dosage, administration, contraindicationsand/or warnings concerning the use of such therapeutic products. Theinstructional materials may be written, in an electronic form (such as acomputer diskette or compact disk) or may be visual (such as videofiles). The kits may also include additional components to facilitatethe particular application for which the kit is designed. Thus, forexample, the kit may additionally contain means of detecting a label(such as enzyme substrates for enzymatic labels, filter sets to detectfluorescent labels, appropriate secondary labels such as a secondaryantibody, or the like). The kits may additionally include buffers andother reagents routinely used for the practice of a particular method.Such kits and appropriate contents are well known to those of skill inthe art.

EXAMPLES

The following examples are provided to illustrate particular features ofcertain embodiments, but the scope of the claims should not be limitedto those features exemplified.

Example 1 Human Monoclonal Antibodies and Fragments Thereof TargetingMatrilin-3 in Growth Plate Cartilage

This example illustrates monoclonal antibodies and antigen bindingfragments thereof for targeting therapeutics to growth plate cartilage.

Abstract

Growth plate function can be severely impaired in many acquired systemicdisorders, and in genetic disorders, including skeletal dysplasias, inwhich the bones are typically short and malformed, causing disability.Current medical therapies have limited efficacy for severe disease, withdoses limited by effects on tissues other than growth plate cartilage.Recent studies have identified many paracrine factors that act in thegrowth plate to positively regulate chondrogenesis, and therefore mightbe used therapeutically if they could be targeted to growth platecartilage. Similarly, targeting growth-promoting endocrine factors, suchas growth hormone and insulin-like growth factor-I, specifically to thegrowth plate could potentially enhance therapeutic efficacy whilediminishing adverse effects on non-target tissues. Using yeast displaytechnology, single-chain variable antibody fragments were identifiedthat bind with high affinity to human and mouse matrilin-3, a proteinexpressed specifically in cartilage tissue. They also bound with highspecificity to cartilage homogenates and to cartilage structures inmouse embryo sections. When injected intravenously in mice, theseantibody fragments homed to cartilage and were not detectable in othertissues. Coupling these cartilage-binding antibodies to endocrine andparacrine signaling molecules that promote chondrogenesis can allowtherapy targeted specifically to growth plate, and also articularcartilage, thereby opening up broad new pharmacological approaches totargeted drug delivery to deliver drugs to the growth plate or articularcartilage, for example to increase height and/or to treat cartilagedisorders, such as skeletal dysplasias, including achondroplasia, shortstature, and joint diseases such as osteoarthritis.

Introduction

The growth plate is a specialized cartilage structure present near theends of tubular bones and vertebrae. The primary function of the growthplate is to generate new cartilage, which is then remodeled into bonetissue, resulting in bone elongation. In this process, termedendochondral bone formation, chondrocytes in the growth plate undergorapid proliferation followed by terminal differentiation intohypertrophic chondrocytes. The newly formed cartilage is invaded byblood vessels and bone cells, which convert the cartilage into bonetissue. The net result is that new bone is formed adjacent to the growthplate, leading to longitudinal bone growth. Because body length islargely determined by the lengths of long bones and vertebrae,endochondral bone formation at the growth plates is the underlyingmechanism responsible for increasing height during childhood.

Longitudinal bone growth is a complex process which requires multipleintracellular, endocrine, and paracrine pathways to function normally.Consequently, mutations in hundreds of genes that are required forgrowth plate function give rise to disorders of skeletal growth,including the skeletal dysplasias, in which the bones are typicallyshort and malformed, often causing major disability. In addition togenetic disorders, acquired endocrine, nutritional, or inflammatorydisorders can also impair bone growth at the growth plate, resulting insevere short stature.

Current treatment options for growth disorders are limited. Recombinanthuman growth hormone (GH) is used for both GH-deficiency and certainnon-GH-deficient causes of short stature (Richmond, Current Indicationsfor Growth Hormone Therapy Vol. 18, Karger, 2010). However, the efficacyof GH treatment is often suboptimal. Even for growth hormone deficiency,the reported adult heights achieved in the majority of patients after GHsupplementation remain below the normal range (Ranke, et al. Horm ResPaediatr, 51-67, 2013). In non-GH deficient conditions, includingskeletal dysplasias, the efficacy is typically even more partial (Ranke.Pediatrician 14, 178-182, 1987). Moreover, GH treatment carries a riskof increased intracranial pressure (Wilson, et al. J Pediatr 143,415-421, 2003), slipped capital femoral epiphysis (Wang, et al. J FormosMed Assoc 106, S46-50, 2007; and Darendeliler, et al. Horm Res 68 Suppl5, 41-47, 2007), insulin resistance (Yuen, et al. Diabet Med, 30.6,651-663, 2013; and Canete, et al. Eur J Endocrinol 167, 255-260, 2012),and possibly type II diabetes mellitus (Cutfield, et al. Lancet 355,610-613, 2000). Endogenous GH excess increases the risk of colon cancer;whether or not childhood growth hormone treatment raises cancer risk inadulthood is not known (Swerdlow, et al. Lancet 360, 273-277, 2002).Because systemic administration of GH has limited efficacy andsignificant known and potential adverse effects, better treatments forgrowth plate disorders are needed.

Recent studies have identified many paracrine factors that positivelyregulate growth plate chondrogenesis and therefore might be usedtherapeutically, including Indian Hedgehog (IHH) (Chau, et al. J MolEndocrinol 47, 99-107, 2011; Kobayashi, et al. J Clin Invest 115,1734-1742, 2005; Kronenberg. Ann N Y Acad Sci 1068, 1-13, 2006; Maeda,et al. Proc Natl Acad Sci USA 104, 6382-6387, 2007; Long, et al. DevBiol 298, 327-333, 2006; Amizuka, et al. J Cell Biol 126, 1611-1623,1994; Long, et al. Development 128, 5099-5108, 2001; Mak, et al.Development 135, 1947-1956, 2008), bone morphogenetic proteins (BMPs)(De Luca, et al. Endocrinology 142, 430-436, 2001; Nilsson, et al. JEndocrinol 193, 75-84, 2007; Kobayashi, et al. Proc Natl Acad Sci USA102, 18023-18027, 2005; Yoon, et al. Development 133, 4667-4678, 2006;Wu, et al. J Biol Chem 286, 24726-24734, 2011; Yoon, et al. Proc NatlAcad Sci USA 102, 5062-5067, 2005), C-type natriuretic peptide (CNP)(Mericq, et al. Pediatr Res 47, 189-193, 2000; Agoston, et al. BMC DevBiol 7, 18, 2007; Olney, et al. J Clin Endocrinol Metab 92, 4294-4298,2007; Olney, et al. J Clin Endocrinol Metab 91, 1229-1232, 2006;Teixeira, et al. Dev Biol 319, 171-178, (2008); Woods, et al.Endocrinology 148, 5030-5041, 2007), and WNTs (Andrade, et al. Bone 40,1361-1369, 2007; Hartmann, et al. Development 127, 3141-3159, 2000;Yates, et al. DNA Cell Biol 24, 446-457, 2005; Yang, et al. Development130, 1003-1015, 2003; Akiyama, et al., Genes Dev 18, 1072-1087, 2004).However, the development of these molecules into effective treatment hasbeen hampered by their mechanism of action; these growth factors areproduced locally and act locally in the growth plate, and thus do notlend themselves to systemic therapeutic approaches. Disclosed herein aremethods of targeting these locally-acting molecules to the growth plateby linking them to cartilage-binding proteins, such as antibodyfragments. When administered systemically, these hybrid molecules wouldbe preferentially taken up by growth plate cartilage, and thus augmentthe therapeutic effect on the target organ while diminishing adverseeffects due to action on other tissues.

Similarly, growth-promoting endocrine factors, such as GH andinsulin-like growth factor-I can be linked to cartilage-bindingpolypeptides and thereby targeted to the growth plate. Targetedendocrine therapy could potentially enhance the therapeutic effects onchondrogenesis or bone growth and reduce effects on non-target tissues,thereby decreasing risks such as malignancy and diabetes mellitus.Besides treating growth plate disorders, cartilage-targetingpolypeptides might also be applied to improve treatment of articularcartilage disorders.

As described below, proteins that home to cartilage tissue wereidentified. A yeast-displayed library of human antibody fragments wasscreened for high affinity binders to matrilin-3, a protein expressedwith high tissue specificity in cartilage. Several antibody fragmentswere identified that bind with high affinity both to human and mousematrilin-3, as well as to cartilage tissue in vitro. When these antibodyfragments were administered to mice by tail-vein injection, they homedspecifically to cartilage tissue. Coupling these antibody fragments toendocrine and paracrine factors that stimulate chondrogenesis could beused to direct these potent molecules specifically to cartilage tissue,providing important new therapeutic approaches to the treatment ofgrowth plate and articular cartilage disorders.

Material and Methods Selection of Extracellular Matrix Protein-BindingAntibody Fragments

Recombinant human and mouse matrilin-3 proteins (R&D Systems,Minneapolis, MN) that were biotinylated using an Avi-tag SpecificBiotinylation kit (Aurora, CO) were used as the target for selection. Inthe first round of selection, approximately 5×10¹⁰ cells from the naïveantibody library were incubated with 10 μg of biotinylated humanmatrilin-3 in 50 ml 0.1% bovine serum albumin (BSA)-phosphate-bufferedsaline (PBS), called PBSA, at room temperature for two hours with gentlerotation. Then, the mixture was washed three times with 0.1% PBSA toremove unbound antibody fragments. Biotinylated matrilin-3 together withbound antibody fragments were subsequently incubated with 100 μl ofstrepatavidin-conjugated microbeads (Milenvi Biotec, Auburn, CA) andloaded onto the AutoMACS system for sorting. Cells which displayantibody fragments with high affinity to matrilin-3 were collected andlater amplified in SDCAA Medium (20 g Dextrose, 6.7 g Difco yeastnitrogen base without amino acids, 5 g Bacto casamino acids, 5.4 gNa₂HPO₄ and 8.56 g NaH₂PO₄. H₂O dissolved in 1 L of distilled water) at250 rpm at 30° C. for 24 hours. After that, the culture was induced inSGCAA Medium (20 g Galactose, 20 g Raffinose, 1 g Dextrose, 6.7 g Difcoyeast nitrogen base without amino acids, 5 g Bacto casamino acids, 5.4 gNa₂HPO₄ and 8.56 g NaH₂PO₄. H₂O dissolved in 1 L of distilled water) at250 rpm at 20° C. for 18 hours. The pool obtained was subjected toanother round of selection for binding to human matrilin-3. To ensuresufficient diversity of antibody fragments for second and third roundsof screening, the input cell number was increased by 100 fold comparedto the prior round of sorting.

For the third round of selection, His-Tagged recombinant mousematrilin-3 was employed. The screening was carried out in a similar wayto the previous two rounds of selection toward human matrilin-3.Finally, antibody fragments that bound to mouse matrilin-3 were pulleddown by anti-His-tag antibody-conjugated microbeads. The yeast cellsexpressing antibody fragments that possess high binding affinity tohuman and mouse matrilin-3 proteins were collected.

Cloning of Extracellular Matrix Protein-Binding Antibody Fragments inMammalian Vectors

After the final round of sorting, DNA plasmids were extracted from theyeast cells using Yeast Plasmid Extraction kit (Zymo Research, Irvine,CA) and then transformed into 10G chemical competent E. coli (Lucigen,Middleton, WI) for further amplification. The scFv-encoding DNA insertswere double-digested with restriction enzyme SfiI and ligated to amodified pSecTagB vector, which bears the same set of SfiI sites and adownstream Fc-Avi tag.

Cell Culture

HEK293T cells were obtained from ATCC (Manassas, VA), and maintained inDulbecco's modified Eagle's Medium (DMEM) (Gibco, Grand Island, NY)supplemented with 10% fetal calf serum (FCS) (v/v) (Gibco) and 1%penicillin-streptomycin (PS) (v/v) (Gibco) at 37° C. in a humidifiedatmosphere of 5% CO₂. FreeStyle 293-F cells (Invitrogen, Grand Island,NY) were cultured in suspension in FreeStyle 293 Expression Medium(Gibco) shaking at 125 rpm at 37° C. in a humidified atmosphere of 8%CO₂.

Small-Scale Expression of Antibody Fragments in HEK293T Cells

HEK293T cells were seeded in 24-well plates at a density of 5×10⁴cells/well, and cultured at 37° C. in 5% CO₂ overnight. On the next day,0.5 μg of DNA and 5 μl of PolyFect reagent (Qiagen, Valencia, CA) weresuspended in serum-free, antibiotic-free DMEM to give a final volume of30 μl, which was allowed to stand at room temperature for 10 minutes.Then, the mixture was introduced to the wells containing cultured cellsand 375 μl of fresh medium. At 48 hours post-transfection, supernatantcontaining the secreted antibody fragments was collected for subsequentexperiments.

Assessment of the Binding Ability and Specificity of Antibody Fragments

To assess the ability of the 36 selected clones of antibody fragments tobind to purified matrilin-3, 100 μl of human or mouse matrilin-3 protein(2 μg/ml) was coated onto 96-well plates at 4° C. overnight. To assessbinding to cartilage and non-cartilaginous tissues, heart, liver, lung,kidney, spleen, small intestine, muscle, and distal femoral and proximaltibial growth plate were dissected from 4-day-old C57BL/6 mice andhomogenized in protein lysis buffer (150 mM NaCl, 10 mM Tris-HCl, 5 mMEDTA (pH8.0), 1% Triton X-100, 0.1% SDS) at 4° C. Tissue debris wasremoved by centrifugation. 100 μl of tissue lysate was coated onto96-well plates at 4° C. overnight. Each antibody clone was tested intriplicate wells. After blocking with 3% non-fat milk (200 μl/well), 50μl of the culture supernatant was introduced to each well and incubatedat room temperature for two hours. Supernatant from cells transfectedwith a non-specific antibody fragment was included as a negativecontrol, while commercial anti-matrilin-3 antibody polyclonal antibodyrecognizing a 13-amino acid peptide from near the center of humanmatrilin-3 (Thermo Scientific, Rockford, IL) served as a positivecontrol. The wells were then washed with 0.05% Tween-phosphate-bufferedsaline (PBST) four times and incubated with 50 μl of horseradishperoxidase (HRP)-conjugated anti-Fc antibody (Millipore, Temecula, CA)(diluted 1:5000 in 3% non-fat milk) at room temperature for one hour.Finally, tetramethybezidine (TMB) substrate reagent (eBioscience, SanDiego, CA) was added for color development and absorbance was read at450 nm.

Examination of Binding of Selected Antibody Fragments in Mouse EmbryoSections

10 μm cryosections of frozen E15 mouse embryos were equilibrated to roomtemperature for 30 minutes, fixed in acetone for 15 minutes, air-driedfor 30 minutes, and then blocked with 1% FCS in PBS at room temperaturefor 1 hour. The sections were then incubated for 1 hour with 100 μl ofthe supernatant from transfected HEK293T cells expressing antibodyfragments, washed with PBS for 5 minutes three times, incubated for 1hour with 100 μl of HRP-conjugated anti-Fc antibody (1:2000 dilution inPBS containing 1% FCS), and washed with PBS for 5 minutes three times,all at room temperature. Binding of the selected antibody fragments tosections was detected using DAB substrate kit (Abcam, Cambridge, MA). Anantibody fragment selected for binding to a protein of Dengue virusserved as a negative control.

Large-Scale Expression of Selected Antibody Fragments in 293 FreeStyle-FCells

For production and purification, antibody fragments were expressed in293 FreeStyle-F, a suspension cell line which is adapted to serum-freemedium and thus avoids serum IgG which may interfere with antibodyfragment purification. Twenty-four hours before transfection, 3×10⁷ 293FreeStyle-F cells were resuspended in 28 ml FreeStyle 293 ExpressionMedium in a 250 ml Erlenmeyer flask on an orbital shaker rotating at 125rpm, at 37° C. in 8% CO₂. On the day of transfection, 30 μg of DNA and60 μl of 293fectin reagent (Qiagen) were diluted in 2 ml of Opti-MEM,and the mixture was incubated at room temperature for 20 minutes.Afterward, the mixture was introduced to a flask containing the 293FreeStyle-F cells in 28 ml medium and shaken for 3 days to expresssoluble protein fragments. Supernatant was subsequently collected forantibody purification using protein A columns

Purification of Antibody Fragments by Protein a Column

Protein A resin (GenScript, Piscataway, NJ) slurry (2 ml) was packedinto a glass column, and equilibrated with 50 ml of binding/washingbuffer (0.15 M NaCl, 20 mM Na₂HPO₄, pH 8.0). Culture supernatant wasloaded onto the column. Unbound proteins were washed away with 100 ml ofbinding/washing buffer. Bound antibodies were then eluted with 8 ml ofelution buffer (100 mM acetic acid, pH 3.0). The eluate was neutralizedby 1/10 volume of neutralization buffer (1 M Tris-HCl, pH 9.0) anddialyzed against 100 volumes of PBS at 4° C. overnight. The purity ofthe antibodies was checked by SDS-PAGE.

Measurement of the Binding Affinity of Antibody Fragments

To assess the binding affinity of the purified antibody fragments, 100μl of human or mouse matrilin-3 protein (2 μg/ml) was coated onto96-well plates at 4° C. overnight. Then, the wells were blocked with 200μl of 3% non-fat milk in PBS at room temperature for 2 hours. 50 μl ofdifferent concentrations of antibody fragments 13, 22, and 26 (230 nM,57.5 nM, 14.375 nM, 3.594 nM, 0.899 nM, 0.225 nM, 0.056 nM, 0.014 nM)was added to incubate with the target at room temperature for 2 hours.Duplicates were performed for each concentration of the selectedantibody fragments. After binding, the wells were washed with 0.05% PBST4 times and incubated with 50 μl of HRP-conjugated anti-Fc antibody(diluted 1:5000 in 3% non-fat milk) at room temperature for 1 hour.Lastly, TMB substrate reagent was introduced and absorbance was read at450 nm.

Investigation of the In Vivo Homing Ability of Antibody Fragments

In order to evaluate whether the selected antibodies were able to hometo growth plate cartilage, 50 μg of purified antibody fragments, dilutedin 100 μl of sterile saline, was injected in 3-week-old C57BL/6 malemice intravenously. As control experiments, two groups of mice wereinjected with 100 μl of sterile saline or 50 μg (in 100 μl of sterilesaline) of purified non-specific antibody fragment (selected for bindingto a protein of Dengue virus). After 24 hours, tissues were harvestedand homogenized in protein lysis buffer. Tissue debris was removed bycentrifugation, and supernatant was collected for ELISA to check for thelocalization of the antibody fragments. Briefly, 100 μl of tissue lysatewas coated in triplicate wells in 96-well plates at 4° C. overnight.Subsequent to blocking with 200 μl of 3% non-fat milk per well, 50 μl ofHRP-conjugated anti-Fc antibody (diluted 1:5000 in 3% non-fat milk) wasadded to each well and incubated at room temperature for 1 hour. TMBsubstrate reagent was then introduced and the absorbance was read at 450nm. For each organ or tissue in a particular group, statisticalsignificance was assessed by ANOVA, followed by pairwise comparison thegrowth plate signal to other tissues, with Holm-Sidak correction formultiple comparisons. Comparison of the growth plate signal betweendifferent groups were done by Student t-test.

Results Selection of Extracellular Matrix Protein-Specific AntibodyFragments

Antibody fragments were selected from a large yeast display single-chainfragment variable (scFv) library for binding to matrilin-3, anextracellular matrix protein specifically expressed in cartilage(Wagener, et al. FEBS Lett 413, 129-134, 1997; Burnam, et al. J AbnormPsychol 84, 76-79, 1975; Klatt, et al. J Biol Chem 275, 3999-4006,2000). The sorting was carried out against human recombinant matrilin-3protein in the first two rounds and mouse recombinant matrilin-3 proteinin the third round of panning to increase the likelihood that theresulting antibody fragments would bind both to mouse cartilage matrixfor preclinical safety and efficacy evaluations and human cartilagematrix, for clinical applications. After three rounds of selection, theenriched library exhibited a striking increase in binding to matrilin-3proteins of both species by flow cytometric analysis (FIG. 1A).

Binding of Antibody Fragments to Cartilage Matrix In Vitro

After the final round of selection, 36 yeast clones were randomlyselected and the expression plasmids were isolated. The DNA sequencesencoding the scFvs were then excised and subcloned into a mammalianexpression vector pSecTagB, which was previously engineered to include ahuman Fc fragment-coding DNA sequence. The resulting construct consistedof a scFv antibody fragment fused with human IgG1 Fc fragment. Thisconstruct was subsequently transfected into HEK293T cells for 48 hoursto express scFv-Fc proteins, and the ability of these 36 proteins tobind both human and mouse recombinant matrilin-3 was assessed by ELISA.Of these, 21 bound to human recombinant matrilin-3 (FIG. 1B), and 17bound to mouse matrilin-3 (FIG. 1C), compared to bovine serum albuminand to a non-specific antibody fragment.

Binding of Antibody Fragments to Cartilage Extracts In Vitro

The selected antibody fragments were tested for tissue-specific bindingto cartilage, where matrilin-3 is predominantly expressed (Wagener, etal. FEBS Lett 413, 129-134, 1997; Burnam, et al. J Abnorm Psychol 84,76-79, 1975; Klatt, et al. J Biol Chem 275, 3999-4006, 2000). To examinethe tissue specificity of the antibody fragments, homogenized tissue wasused from growth plate cartilage, brain, heart, liver, lung, kidney,spleen, small intestine, and muscle from 4-day old mice to coat plasticwells and the binding of the antibody fragments was assessed by ELISA.Fifteen antibody fragments that exhibited increased binding tomatrilin-3 of both species were examined for their specificity towardcartilage tissue. While some antibody fragments demonstrated poor tissuebinding specificity, antibody fragments 13, 22, and 26 were found topreferentially bind to growth plate cartilage over other tissues (FIG.1D), and thus were chosen for further studies. A non-specific antibodyfragment showed binding to cartilage or other tissues.

Immunohistochemical Localization of Matrilin-3-Binding AntibodyFragments

To further characterize the ability and specificity of the antibodyfragments to bind to cartilage tissue, the antibody fragments were usedto perform immunohistochemical staining on frozen sections of C57BL/6mouse embryos at embryonic day (E) 15. The three antibody fragments (13,22 and 26) stained various cartilage structures, such as thecartilaginous anlagen of the bones of the forelimb, but there was nosignificant staining in non-cartilaginous tissues (FIGS. 2A-C). Incontrast, a non-specific antibody fragment failed to show any stainingin the cartilage tissues.

Assessment of the Binding Affinity of Purified Matrilin-3-BindingAntibody Fragments

To measure binding affinity quantitatively, different concentrations ofantibody fragments 13, 22, and 26 were used to test binding tomatrilin-3 proteins, both human and mouse. All three antibody fragmentspossessed high affinity towards both recombinant human and mousematrilin-3 proteins with EC₅₀ values less than 1 nM (FIGS. 3A and 3B).

Homing of Matrilin-3-Binding Antibody Fragments to Cartilage In Vivo

Purified antibody fragments were injected into mice via a tail vein.After 24 hours, distal femoral and proximal tibial growth platecartilage and various non-cartilaginous organs were isolated,homogenized and used to coat plastic wells. ELISA was performed usinganti-human Fc antibody to detect the presence of antibody fragmentsdelivered in vivo. For both the saline-injected and the non-specificantibody fragment-injected controls, the signals in the growth plate andother non-cartilaginous organs were similar. In contrast, for the threeantibody fragments tested (13, 22 and 26), the signals for saline or fornon-specific antibody fragments (FIG. 4 ). Furthermore, for these threeantibody fragments, the signals were significantly greater in cartilagethan in non-cartilaginous organs (FIG. 4 ), suggesting that the selectedantibody fragments homed to cartilage in vivo with high specificity.

Discussion

Antibody fragments that bind to cartilage with high affinity andspecificity were identified, and can be used to target therapeuticmolecules to growth plate cartilage. A naïve human scFv yeast displaylibrary was used for selection of binders to matrilin-3, anextracellular matrix protein primarily expressed in growth platecartilage. A sequential antigen panning approach (Zhang, et al. JImmunol Methods 283, 17-25, 2003) was used, with the first two rounds ofpanning against human matrilin-3 and the last round against mousematrilin-3 protein. The resulting pool of clones was significantlyenriched for binders to both human and mouse matrilin-3, compared to thenaïve library. Individual clones that expressed antibody fragments thatbound to both human and mouse matrilin-3 were identified. Of these,three antibody fragments showed specific binding in vitro to homogenatesof cartilage tissue, but not homogenates of brain, heart, liver, lung,kidney, spleen, small intestine or muscle. These three fragments alsoshowed tissue-specific binding to cartilage structures in sections ofmouse embryos. Binding affinities of the selected antibody fragments(12, 22, and 26) were then measured, demonstrating that all threepurified antibody fragments exhibited high affinity for both human andmouse matrilin-3 proteins. After these purified antibody fragments wereinjected intravenously in mice it was found that they were localized incartilage and were not detectable in other tissues, including brain,heart, liver, lung, kidney, spleen, small intestine or muscle,indicating that the antibody fragments were capable of specificallytargeting cartilage tissue in vivo.

The development of cartilage-targeting proteins opens up new potentialapproaches to treat growth plate disorders, including skeletaldysplasias, severe short stature due to systemic disease, and severeidiopathic short stature, by targeting growth-regulating endocrinefactors specifically to the growth plate. Current growth plate therapygenerally involves the manipulation of systemic hormone levels, such asGH, IGF-1, estrogens, and androgens. However, these approaches havelimited therapeutic efficacy for the more severe growth plate disorders,including many skeletal dysplasias, and exhibit undesirable effects dueto actions on sites other than the growth plate. For instance, inachondroplasia, the most common type of skeletal dysplasia, growthhormone increases bone length, and therefore height, only modestly, anddose is limited by adverse effects on other tissues (Horton, et al.Lancet 370, 162-172, 2007). Coupling growth-regulating endocrine factorsto cartilage-binding antibody fragments has the potential to directendocrine therapeutic agents to cartilage. The results presented hereinsuggest that when administered systemically, the targeting antibodyfragment-endocrine factor conjugates would be preferentially taken up bythe growth plate cartilage, thereby creating a local depot which mightallow sustained high local concentrations of molecules to improveefficacy at the growth plate and decrease adverse effects on othertissues.

In addition to endocrine factors, paracrine factors that stimulategrowth plate chondrogenesis, including IHH (Chau, et al. J MolEndocrinol 47, 99-107, 2011; Kobayashi, et al. J Clin Invest 115,1734-1742, 2005; Kronenberg. Ann N Y Acad Sci 1068, 1-13, 2006; Maeda,et al. Proc Natl Acad Sci USA 104, 6382-6387, 2007; Long, et al. DevBiol 298, 327-333, 2006; Amizuka, et al. J Cell Biol 126, 1611-1623,1994; Long, et al. Development 128, 5099-5108, 2001; Mak, et al.Development 135, 1947-1956, 2008), BMPs (De Luca, et al. Endocrinology142, 430-436, 2001; Nilsson, et al. J Endocrinol 193, 75-84, 2007;Kobayashi, et al. Proc Natl Acad Sci USA 102, 18023-18027, 2005; Yoon,et al. Development 133, 4667-4678, 2006; Wu, et al. J Biol Chem 286,24726-24734, 2011; Yoon, et al. Proc Natl Acad Sci USA 102, 5062-5067,2005), and CNP (Mericq, et al. Pediatr Res 47, 189-193, 2000; Agoston,et al. BMC Dev Biol 7, 18, 2007; Olney, et al. J Clin Endocrinol Metab92, 4294-4298, 2007; Olney, et al. J Clin Endocrinol Metab 91,1229-1232, 2006; Teixeira, et al. Dev Biol 319, 171-178, (2008); Woods,et al. Endocrinology 148, 5030-5041, 2007), can be coupled to growthplate targeting antibody fragments. Because these paracrine factors arenormally expressed and exert their action in the growth plate, targetedtherapy would serve to localize these factors to their physiologicalsite of action. For example, CNP is an important positive regulator ofgrowth plate chondrogenesis (Mericq, et al. Pediatr Res 47, 189-193,2000; Agoston, et al. BMC Dev Biol 7, 18, 2007; Olney, et al. J ClinEndocrinol Metab 92, 4294-4298, 2007; Olney, et al. J Clin EndocrinolMetab 91, 1229-1232, 2006; Teixeira, et al. Dev Biol 319, 171-178,(2008); Woods, et al. Endocrinology 148, 5030-5041, 2007). In mice,overexpression of CNP in growth plate compensates for mutations thatcause achondroplasia (Yasoda, et al. Nat Med 10, 80-86, 2004). However,systemic administration of CNP in humans leads to natriuresis (Igaki, etal. Hypertens Res 21, 7-13, 1998). CNP linked to a cartilage-targetingpolypeptide is believed to increase the skeletal growth-promoting effectand reduce the effect on renal sodium handling, and thereby presents aneffective therapeutic strategy to treat human achondroplasia. Similarly,targeting IHH or BMPs to growth plate cartilage might provide noveltreatments for skeletal dysplasias or other causes of growth platefailure, such as systemic inflammatory diseases, renal failure,glucocorticoid therapy or radiation damage.

Apart from treating growth plate disorders, cartilage-targetingpolypeptides could also be applied to improve treatment of articularcartilage disorders. For instance, osteoarthritis, a disorder caused bygradual mechanical degradation of articular cartilage with inadequaterepair, affects 30-50% of older adults (Loeser. Clin Geriatr Med 26,371-386, 2010). Currently, there is no specific pharmacologic therapythat can prevent the progression of joint damage due to osteoarthritis.Cartilage-targeting antibody fragments, which direct chondrogenic growthfactors to articular cartilage, can facilitate regeneration of degradingjoint surfaces.

Methods of fusing a chondrogenic agent to the disclosed antibodyfragments can be similar to previously described fusion techniquesinvolving the biologically active polypeptide, a linker, and an antibodyfragment. For example, multiple cytokine-antibody fusion proteinsdesigned to target malignant tissues and therefore enhance efficacy anddiminish systemic side effects are under investigation, including somein clinical trials (Pasche, et al. Drug Discov Today 17, 583-590, 2012).A similar strategy has been employed to link alkaline phosphatase topeptides that bind to bone matrix, thereby successfully targeting bonetissue and treating hypophosphatasia in mice and humans (Millan, et al.J Bone Miner Res 23, 777-787, 2008; and Yadav, et al. Bone 49, 250-256,2011). Similar techniques can be used to fuse proteins containing thecartilage matrix-binding antibody fragments and biologically activepolypeptides, such as GH, IGF-1 and CNP. Additionally, steroids, such asestradiol, can be coupled to the disclosed antibodies to treat growthdisorders and/or increase height using known techniques (Yokogawa, etal. Endocrinology 142, 1228-1233, 2001).

There have been prior attempts to develop cartilage-binding peptides orpolypeptides with the specific goal of targeting drugs to articularcartilage, for example to treat articular cartilage disorders. A linearpeptide of six amino acids was reported to bind to collagen Hai anddirect nanoparticles to articular cartilage (Rothenfluh, et al. NatMater 7, 248-254, 2008), when given locally by intraarticular injection.A scFv antibody fragment was identified that recognized reactive oxygenspecies-modified type II collagen, which is specifically present ininflamed joints (Hughes, et al. Arthritis Rheum 62, 1007-1016, 2010).This scFv was then coupled to soluble tumor necrosis factor receptor IIand administered systemically to reduce inflammation of articularcartilage in a murine arthritis model. Because the first study involveslocal joint administration and the second study targets inflamedcartilage, neither would be expected to target growth plate cartilage. Aprior study used a 12-amino acid peptide to target growth platecartilage (Cheung, J. C. Lui, and Baron, J Orthop Res., 31:1053-1058,2013), but no high affinity and specificity for cartilage comparable tothat achieved in the current study using antibody fragments wasidentified.

In conclusion, antibody fragments were identified that bind to cartilagematrix with high affinity and specificity and, when administeredsystemically in vivo, home specifically to cartilage structure. Couplingthese cartilage-binding polypeptides to endocrine and paracrinesignaling molecules that promote chondrogenesis can allow therapytargeted specifically to growth plate and articular cartilage, and thusopen up broad new pharmacological approaches to treat skeletaldysplasias, other severe forms of linear growth failure, and jointdiseases. Similarly, targeting such molecules (such as growth hormone)to the antibody fragments can increase the efficacy of the agent andreduce unwanted side effects at tissues where the biological activity ofthe agent is not desired. For example, growth hormone can be deliveredto the desired site of action at the growth plate while minimizingunwanted activity elsewhere in the body that could lead to tumorgenesis.

Example 2 Matril-3 Specific Antibodies Conjugated to a ChondrogenicAgent

This example illustrates construction of matrilin-3 specific antibodiesthat are conjugated to a chondrogenic agent, IGF-1.

Two approaches were taken to engineer the mAb-IGF-1 conjugates,generating dimeric conjugate and monomeric IGF-1/matrilin-3 scFv/Fcdomain conjugates. First, a wildtype Fc domain was used to construct adimeric fusion protein including two scFv-based matrilin-3 bindingdomains, two Fc domains and two IGF-1 domains (see FIG. 5A). Thewildtype Fc domain forms a dimer and is linked to a single IGF-1molecule and a single matrilin-3 specific scFv. Second, a mutant(non-dimerizing) Fc domain was used to construct an antibody-conjugateincluding a single Fc domain linked to a single scFv-based matrilin-3binding domain and a single IGF-1 molecule (see FIG. 5B). Plasmid DNAexpression vectors were constructed encoding the IGF-1-antibody-Fcconjugates (see FIGS. 5A and 5B).

The order of the domains in each conjugate is as follows:

Dimeric conjugates:IGF-1-linker-matrilin-3 specific scFv (based on clone 13, 22, or26)-linker-dimeric Fc domainMonomeric conjugates:IGF-1-linker-monomeric Fc domain-linker-matrilin-3 specific scFv (basedon clone 13, 22, or 26)

Exemplary amino acid sequences of fusion proteins including IGF-1 linkedto a clone 13-, 22-, or 26-based scFv linked to a native Fc domain(dimeric IGF-1-scFv-Fc conjugates) as shown in FIG. 5A are provided asSEQ ID NOs: 41-43, which include scFvs based on the clone 13, clone 22or clone 26 antibodies, respectively. Exemplary amino acid sequences offusion proteins including IGF-1 linked to a mutant Fc domain linked to aclone 13-, 22-, or 26-based scFv (monomeric IGF-1-Fc-scFv conjugates) asshown in FIG. 5B are provided as SEQ ID NOs: 44-46, which include scFvsbased on the clone 13, clone 22 or clone 26 antibodies, respectively.

The IGF-1-scFv conjugates were produced by expression in 293 FreeStyle Fcells, purified, and tested for binding to lysates from severaldifferent tissue types by ELISA. The conjugates were produces usingsimilar methods s those described for production of antibody alone,above. The ELISA plates were coated with the panel of organ lysateslisted in FIG. 6 and incubated with clone 13, 22, or 26 scFv, or thecorresponding IGF-1-scFv-Fc fusion proteins. The results shown were fromassays using dimeric conjugates. As shown in FIG. 6 , the IGF-1-scFv-Fcconjugates demonstrated binding affinity and specificity towards growthplate cartilage comparable of that of the unconjugated clone 13, 22, and26 scFvs, suggesting that addition of IGF-1 and Fc domains did notsignificantly alter the ability of the scFv domains to recognizematrilin-3.

IGF-1 treatment of MCF-7 cells is known to induce Erk and Aktphosphorylation in the MCF-7 breast cancer cell line (Zhang et al. JMol. Endocrinol., 35:433-447, 2005; Walsh and Damjanovski, Cell Commun.Signal., 9:10, 2011). Therefore, to verify that fusion with a matrilin-3specific scFv does not interfere with the biological activity of IGF-1,the IGF-1-induced Erk and Akt phosphorylation in MCF-7 cells was assayedin the context of IGF-1 alone and the IGF-1-matrilin-3 scFv-Fcconjugates using an established assay (Zhang et al. J Mol. Endocrinol.,35:433-447, 2005; Walsh and Damjanovski, Cell Commun. Signal., 9:10,2011). Briefly, MCF-7 cells were incubated with the IGF-1 alone (10 μM)or IGF-1 conjugated to matrilin-3 specific scFV and Fc domains for 30minutes, lysed, and total Akt and phospho-AKT were measured by Westernblot with Akt-specific and phosph-Akt specific antibodies. As shown inFIG. 7 , there was no substantial difference in phosphorylation of Aktbetween the treatment with IGF-1 alone condition compared to treatmentwith the IGF-1-matrilin specific scFv-Fc fusion proteins. DMSO was usedas solvent control, GAPDH was used as loading control.

Example 3 Clinical Evaluation of Therapeutic Agents

The following example provides guidance on the parameters to be used forthe clinical evaluation of a conjugate including a monoclonal antibodyor matrilin-3 binding fragment thereof that specifically binds tomatrilin-3 linked to a chondrogenic agent (such as a CNP polypeptide) inthe therapeutic methods of the present disclosure. As discussed herein,the conjugate will be used in the treatment of a cartilage disorder,such as short stature or a skeletal dysplasia. Clinical trials will beconducted which will provide an assessment of doses of the conjugate forsafety, pharmacokinetics, and initial response of both surrogate anddefined clinical endpoints. The trial will be conducted for a minimum,but not necessarily limited to, twenty-four weeks to collect sufficientsafety information on about 10 evaluable patients. The initial dose forthe trials will vary from about 0.001 to about 1.0 mg/kg/week, or any ofthe doses described herein. In the event that the initial dose in thisrange does not produce a significant direct clinical benefit, the doseshould be increased within this range or beyond this range as necessary,and maintained for an additional minimal period of, but not necessarilylimited to, 24 weeks to establish safety and to evaluate efficacyfurther.

Measurements of safety will include adverse events, allergic reactions,complete clinical chemistry panel (including kidney and liverfunctions), urinalysis, and complete blood count with differential. Inaddition, other parameters relevant to clinical benefit are monitored.The present example also includes the determination of pharmacokineticparameters of the conjugate, including absorption, distribution,metabolism, excretion, and half-life and bioavailability in the blood.Such analyses will help refine dose to clinical response.

Methods

Patients undergo a baseline medical history and physical exam, and astandard set of clinical laboratory tests (including CBC, Panel 20,CHSO, and UA). The patients are followed closely with weekly visits tothe clinic. The patients return to the clinic for a complete evaluationone week after completing the treatment period. Should dose escalationbe required, the patients follow the same schedule outlined above.Safety is monitored throughout the trial.

Diagnosis and Inclusion Criteria

The patients may be male or female, with a documented diagnosis of acondition to be treated, such as a cartilage disorder. A specificexample of a cartilage disorder is achondroplasia, which may beconfirmed by genetic testing and other evidence of an FGFR-3 mutation ordysfunction. The ideal age range of achondroplasia patients is frominfant (<1 year of age) to pre-adolescent (<13 years of age). A patientis excluded from this study if the patient is pregnant or lactating; hasreceived an investigational drug within 30 days prior to studyenrollment; or has a medical condition, serious intercurrent illness, orother extenuating circumstance that may significantly decrease studycompliance.

Safety

Therapy with the conjugate will be determined to be safe if nosignificant acute or chronic drug reactions occur during the course ofthe study. The longer-term administration of the drug will be determinedto be safe if no significant abnormalities are observed in the clinicalexaminations, clinical labs, or other appropriate studies.

It is understood that every embodiment of the disclosure describedherein may optionally be combined with any one or more of the otherembodiments described herein. It will be apparent that the precisedetails of the methods or compositions described may be varied ormodified without departing from the spirit of the described embodiments.We claim all such modifications and variations that fall within thescope and spirit of the claims below.

We claim:
 1. A method of increasing chondrogenesis in cartilage tissue,comprising: contacting cartilage tissue with a therapeutically effectiveamount of a conjugate comprising a monoclonal antibody or antigenbinding molecule and an effector molecule, under conditions sufficientto form an immune complex, thereby increasing chondrogenesis in thecartilage tissue, and wherein: (a) the monoclonal antibody and antigenbinding fragment comprise: a heavy chain variable region comprising aheavy chain complementarity determining region (HCDR)₁, a HCDR2, and aHCDR3, and a light chain variable region comprising a light chaincomplementarity determining region (LCDR)₁, a LCDR2, and a L-CDR3, ofthe amino acid sequences set forth as one of: (i) SEQ ID NO: 1 and SEQID NO: 2, respectively (clone 13); (ii) SEQ ID NO: 3 and SEQ ID NO: 4,respectively (clone 22); or (iii) SEQ ID NO: 5 and SEQ ID NO: 6,respectively (clone 26); and wherein the monoclonal antibody and antigenbinding molecule specifically binds to matrilin-3; and (b) the effectormolecule is a growth hormone, an insulin-like growth factor (IGF)-1, anIndian hedgehog polypeptide, a bone morphogenetic protein, a C-typenatriuretic protein (CNP), a Wnt protein, or a biologically activefragment of the Wnt protein that induces chondrogenesis, or a steroid.2. The method of claim 1, wherein: (i) the HCDR1, HCDR2, and HCDR3comprise the amino acid sequences set forth as residues 26-33, 51-58,and 97-106 of SEQ ID NO: 1, respectively, and the LCDR1, LCDR2, andLCDR3 comprise the amino acid sequence set forth as residues 27-32,50-52, and 89-97 of SEQ ID NO: 2, respectively; (ii) the HCDR1, HCDR2,and HCDR3 comprise the amino acid sequences set forth as residues 26-33,53-61, and 100-109 of SEQ ID NO: 3, respectively, and the LCDR1, LCDR2,and LCDR3 comprise the amino acid sequence set forth as residues 26-31,49-51, and 88-97 of SEQ ID NO: 4, respectively; or (iii) the HCDR1,HCDR2, and HCDR3 comprise the amino acid sequences set forth as residues26-33, 51-58, 97-108 of SEQ ID NO: 5, respectively, and the LCDR1,LCDR2, and LCDR3 comprise the amino acid sequence set forth as residues26-33, 51-53, and 90-100 of SEQ ID NO: 6, respectively.
 3. The method ofclaim 1, wherein the heavy and light chain variable regions comprise theamino acid sequences set forth as one of: (i) SEQ ID NO: 1 and SEQ IDNO: 2, respectively; (ii) SEQ ID NO: 3 and SEQ ID NO: 4, respectively;or (iii) SEQ ID NO: 5 and SEQ ID NO: 5, respectively.
 4. The method ofclaim 1, wherein the antigen binding molecule is a Fv, a Fab, a F(ab′)₂,an scFv, or an scFV₂.
 5. The method of claim 1, wherein the effectormolecule is the growth hormone, the IGF-1, or the CNP.
 6. The method ofclaim 5, wherein the effector molecule is the growth hormone.
 7. Themethod of claim 5, wherein the effector molecule is the IGF-1.
 8. Themethod of claim 5, wherein the effector molecule is the CNP.
 9. Themethod of claim 1, wherein the subject has a cartilage disorder, shortstature or skeletal dysplasia.
 10. The method of claim 9, wherein thesubject has the short stature.
 11. The method of claim 9, wherein: (a)the subject has the short stature and wherein the short stature isidiopathic short stature, short stature due to systemic disease, shortstature associated with a dysmorphic syndrome, short stature due tochromosomal abnormalities, iatrogenic short stature, short stature dueto radiation or medications, short stature in children born small forgestational age, or short stature due to undernutrition; or (b) thesubject has the skeletal dysplasia and wherein the skeletal dysplasia isachondroplasia, hypochondroplasia, or short stature homeobox gene (SHOX)deficiency.
 12. A method of targeting an effector molecule to cartilagetissue in a subject, comprising: administering to the subject atherapeutically effective amount of a conjugate comprising a monoclonalantibody or antigen binding molecule, and an effector molecule, underconditions sufficient to form an immune complex, wherein formation ofthe immune complex targets the heterologous effector molecule to thecartilage tissue in the subject, and wherein: (a) the monoclonalantibody and antigen binding fragment comprise: a heavy chain variableregion comprising a heavy chain complementarity determining region(HCDR)₁, a HCDR2, and a HCDR3, and a light chain variable regioncomprising a light chain complementarity determining region (LCDR)₁, aLCDR2, and a L-CDR3, of the amino acid sequences set forth as one of:(i) SEQ ID NO: 1 and SEQ ID NO: 2, respectively (clone 13); (ii) SEQ IDNO: 3 and SEQ ID NO: 4, respectively (clone 22); or (iii) SEQ ID NO: 5and SEQ ID NO: 6, respectively (clone 26); and wherein the monoclonalantibody and antigen binding molecule specifically binds to matrilin-3;and (b) the effector molecule is a growth hormone, an insulin-likegrowth factor (IGF)-1, an Indian hedgehog polypeptide, a bonemorphogenetic protein, a C-type natriuretic protein (CNP), a Wntprotein, or a biologically active fragment of the Wnt protein thatinduces chondrogenesis, or a steroid.
 13. The method of claim 12,wherein: (i) the HCDR1, HCDR2, and HCDR3 comprise the amino acidsequences set forth as residues 26-33, 51-58, and 97-106 of SEQ ID NO:1, respectively, and the LCDR1, LCDR2, and LCDR3 comprise the amino acidsequence set forth as residues 27-32, 50-52, and 89-97 of SEQ ID NO: 2,respectively; (ii) the HCDR1, HCDR2, and HCDR3 comprise the amino acidsequences set forth as residues 26-33, 53-61, and 100-109 of SEQ ID NO:3, respectively, and the LCDR1, LCDR2, and LCDR3 comprise the amino acidsequence set forth as residues 26-31, 49-51, and 88-97 of SEQ ID NO: 4,respectively; or (iii) the HCDR1, HCDR2, and HCDR3 comprise the aminoacid sequences set forth as residues 26-33, 51-58, 97-108 of SEQ ID NO:5, respectively, and the LCDR1, LCDR2, and LCDR3 comprise the amino acidsequence set forth as residues 26-33, 51-53, and 90-100 of SEQ ID NO: 6,respectively.
 14. The method of claim 12, wherein the effector moleculeis the growth hormone, the IGF-1, or the CNP.
 15. The method of claim14, wherein the effector molecule is the growth hormone.
 16. The methodof claim 15, wherein the effector molecule is the IGF-1.
 17. The methodof claim 15, wherein the effector molecule is the CNP.
 18. The method ofclaim 12, wherein the subject has a cartilage disorder, short stature orskeletal dysplasia.
 19. The method of claim 18, wherein the subject hasthe short stature.
 20. The method of claim 18, wherein: (a) the subjecthas the short stature and wherein the short stature is idiopathic shortstature, short stature due to systemic disease, short stature associatedwith a dysmorphic syndrome, short stature due to chromosomalabnormalities, iatrogenic short stature, short stature due to radiationor medications, short stature in children born small for gestationalage, or short stature due to undernutrition; or (b) the subject has theskeletal dysplasia and wherein the skeletal dysplasia is achondroplasia,hypochondroplasia, or short stature homeobox gene (SHOX) deficiency.